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Transcript of -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI...
![Page 1: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/1.jpg)
-Techniques in Glycobiology-NHLBI CardioPEG ndash Gerald WHart August 19 2013
Funded by NHLBI P01HL107153
Introduction to
GlycanAnalysis
Techniques in Glycobiology
2
Topics Overview of Glycoproteomic and Glycomic Approaches
Composition Analysis of Glycans ndash Labs 821 amp 94
Radiochemical and Bioorthogonal Labelling of Glycans ndash Lab 830
Detection of Glycoproteins in Polyacrylamide Gels
Lectin Arrays to Analyze Glycoproteins ndash Lab 1011
Antibody Detection of Specific Glycans in Gels
Detection of Specific Glyosphingolipids on TLC- Lab 107 amp109
Methods for Isolation of Glycoproteins
Enzymatic and Chemical Release of Glycans from Glycoproteins ndash Lab 823
Labeling Released Glycans ndash Lab 830
Methods to Separate Released Glycans ndashLab 911
Glycosidases amp Structural Analyses ndash Lab 918
Methylation Analysis for Linkage Determination ndash Lab 826
MALDI-TOF Profiling of Glycans ndash Lab 1014
Nuclear Magnetic Resonance Spectroscopy ndash Labs 1016 amp 1018
GPI-Anchors amp Glycosaminoglycans ndash Lab 925
Glycosyltransferases As Probes- Lab 9273
J Mass Spectrom 2013 48 627ndash639
Types of Glycoproteomics ndash Require Different Methods
4
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Many Options for Glycoprotein Analyses
5
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Level of Analysis amp Approach is Determined by Biological Question
6
Rough Estimates of Glycans by Colorimetric Assays (Lab 1)
Wed August 21 2013
Phenol-Sulfuric Acid Assay for hexoses amp pentoses
MBTH Assay for Hexosamines amp Acetylhexosamines
Carbazole Assay for Uronic Acids
BCA Assay for Reducing Sugars
Analysis of Free Sialic Acids From Glycoconjugates
by the Thiobarbituric acid assay
7
Useful as a starting place to estimate the quantity of glycans in a tissue or cell
Compositional Analysis by HPAEC-PAD
8
HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection
Compositional Analysis by GC-MS of Alditol Acetates
9
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS
Alditols 1 peak per monosaccharide
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 2: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/2.jpg)
Techniques in Glycobiology
2
Topics Overview of Glycoproteomic and Glycomic Approaches
Composition Analysis of Glycans ndash Labs 821 amp 94
Radiochemical and Bioorthogonal Labelling of Glycans ndash Lab 830
Detection of Glycoproteins in Polyacrylamide Gels
Lectin Arrays to Analyze Glycoproteins ndash Lab 1011
Antibody Detection of Specific Glycans in Gels
Detection of Specific Glyosphingolipids on TLC- Lab 107 amp109
Methods for Isolation of Glycoproteins
Enzymatic and Chemical Release of Glycans from Glycoproteins ndash Lab 823
Labeling Released Glycans ndash Lab 830
Methods to Separate Released Glycans ndashLab 911
Glycosidases amp Structural Analyses ndash Lab 918
Methylation Analysis for Linkage Determination ndash Lab 826
MALDI-TOF Profiling of Glycans ndash Lab 1014
Nuclear Magnetic Resonance Spectroscopy ndash Labs 1016 amp 1018
GPI-Anchors amp Glycosaminoglycans ndash Lab 925
Glycosyltransferases As Probes- Lab 9273
J Mass Spectrom 2013 48 627ndash639
Types of Glycoproteomics ndash Require Different Methods
4
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Many Options for Glycoprotein Analyses
5
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Level of Analysis amp Approach is Determined by Biological Question
6
Rough Estimates of Glycans by Colorimetric Assays (Lab 1)
Wed August 21 2013
Phenol-Sulfuric Acid Assay for hexoses amp pentoses
MBTH Assay for Hexosamines amp Acetylhexosamines
Carbazole Assay for Uronic Acids
BCA Assay for Reducing Sugars
Analysis of Free Sialic Acids From Glycoconjugates
by the Thiobarbituric acid assay
7
Useful as a starting place to estimate the quantity of glycans in a tissue or cell
Compositional Analysis by HPAEC-PAD
8
HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection
Compositional Analysis by GC-MS of Alditol Acetates
9
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS
Alditols 1 peak per monosaccharide
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 3: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/3.jpg)
Topics Overview of Glycoproteomic and Glycomic Approaches
Composition Analysis of Glycans ndash Labs 821 amp 94
Radiochemical and Bioorthogonal Labelling of Glycans ndash Lab 830
Detection of Glycoproteins in Polyacrylamide Gels
Lectin Arrays to Analyze Glycoproteins ndash Lab 1011
Antibody Detection of Specific Glycans in Gels
Detection of Specific Glyosphingolipids on TLC- Lab 107 amp109
Methods for Isolation of Glycoproteins
Enzymatic and Chemical Release of Glycans from Glycoproteins ndash Lab 823
Labeling Released Glycans ndash Lab 830
Methods to Separate Released Glycans ndashLab 911
Glycosidases amp Structural Analyses ndash Lab 918
Methylation Analysis for Linkage Determination ndash Lab 826
MALDI-TOF Profiling of Glycans ndash Lab 1014
Nuclear Magnetic Resonance Spectroscopy ndash Labs 1016 amp 1018
GPI-Anchors amp Glycosaminoglycans ndash Lab 925
Glycosyltransferases As Probes- Lab 9273
J Mass Spectrom 2013 48 627ndash639
Types of Glycoproteomics ndash Require Different Methods
4
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Many Options for Glycoprotein Analyses
5
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Level of Analysis amp Approach is Determined by Biological Question
6
Rough Estimates of Glycans by Colorimetric Assays (Lab 1)
Wed August 21 2013
Phenol-Sulfuric Acid Assay for hexoses amp pentoses
MBTH Assay for Hexosamines amp Acetylhexosamines
Carbazole Assay for Uronic Acids
BCA Assay for Reducing Sugars
Analysis of Free Sialic Acids From Glycoconjugates
by the Thiobarbituric acid assay
7
Useful as a starting place to estimate the quantity of glycans in a tissue or cell
Compositional Analysis by HPAEC-PAD
8
HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection
Compositional Analysis by GC-MS of Alditol Acetates
9
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS
Alditols 1 peak per monosaccharide
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 4: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/4.jpg)
J Mass Spectrom 2013 48 627ndash639
Types of Glycoproteomics ndash Require Different Methods
4
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Many Options for Glycoprotein Analyses
5
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Level of Analysis amp Approach is Determined by Biological Question
6
Rough Estimates of Glycans by Colorimetric Assays (Lab 1)
Wed August 21 2013
Phenol-Sulfuric Acid Assay for hexoses amp pentoses
MBTH Assay for Hexosamines amp Acetylhexosamines
Carbazole Assay for Uronic Acids
BCA Assay for Reducing Sugars
Analysis of Free Sialic Acids From Glycoconjugates
by the Thiobarbituric acid assay
7
Useful as a starting place to estimate the quantity of glycans in a tissue or cell
Compositional Analysis by HPAEC-PAD
8
HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection
Compositional Analysis by GC-MS of Alditol Acetates
9
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS
Alditols 1 peak per monosaccharide
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 5: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/5.jpg)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Many Options for Glycoprotein Analyses
5
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Level of Analysis amp Approach is Determined by Biological Question
6
Rough Estimates of Glycans by Colorimetric Assays (Lab 1)
Wed August 21 2013
Phenol-Sulfuric Acid Assay for hexoses amp pentoses
MBTH Assay for Hexosamines amp Acetylhexosamines
Carbazole Assay for Uronic Acids
BCA Assay for Reducing Sugars
Analysis of Free Sialic Acids From Glycoconjugates
by the Thiobarbituric acid assay
7
Useful as a starting place to estimate the quantity of glycans in a tissue or cell
Compositional Analysis by HPAEC-PAD
8
HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection
Compositional Analysis by GC-MS of Alditol Acetates
9
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS
Alditols 1 peak per monosaccharide
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 6: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/6.jpg)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
Level of Analysis amp Approach is Determined by Biological Question
6
Rough Estimates of Glycans by Colorimetric Assays (Lab 1)
Wed August 21 2013
Phenol-Sulfuric Acid Assay for hexoses amp pentoses
MBTH Assay for Hexosamines amp Acetylhexosamines
Carbazole Assay for Uronic Acids
BCA Assay for Reducing Sugars
Analysis of Free Sialic Acids From Glycoconjugates
by the Thiobarbituric acid assay
7
Useful as a starting place to estimate the quantity of glycans in a tissue or cell
Compositional Analysis by HPAEC-PAD
8
HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection
Compositional Analysis by GC-MS of Alditol Acetates
9
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS
Alditols 1 peak per monosaccharide
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 7: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/7.jpg)
Rough Estimates of Glycans by Colorimetric Assays (Lab 1)
Wed August 21 2013
Phenol-Sulfuric Acid Assay for hexoses amp pentoses
MBTH Assay for Hexosamines amp Acetylhexosamines
Carbazole Assay for Uronic Acids
BCA Assay for Reducing Sugars
Analysis of Free Sialic Acids From Glycoconjugates
by the Thiobarbituric acid assay
7
Useful as a starting place to estimate the quantity of glycans in a tissue or cell
Compositional Analysis by HPAEC-PAD
8
HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection
Compositional Analysis by GC-MS of Alditol Acetates
9
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS
Alditols 1 peak per monosaccharide
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 8: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/8.jpg)
Compositional Analysis by HPAEC-PAD
8
HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection
Compositional Analysis by GC-MS of Alditol Acetates
9
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS
Alditols 1 peak per monosaccharide
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 9: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/9.jpg)
Compositional Analysis by GC-MS of Alditol Acetates
9
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS
Alditols 1 peak per monosaccharide
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 10: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/10.jpg)
Chapter 47 Figure 1
Radiolabeling strategies for the detection of glycans
Essentials of Glycobiology Second Edition
10
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 11: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/11.jpg)
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811
Nice discussion of issues
1 Competition with glucose ndash need to compromise
bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis
bull cells must not exhaust the supply of Glc or other nutrients in the medium
bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation
11
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 12: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/12.jpg)
Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS
12
Analysis of glycans using the bioorthogonal chemical reporter strategy
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 13: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/13.jpg)
Chapter 48 Figure 3
Metabolic and covalent labeling of glycans for in vivo imaging of the glycome
Essentials of Glycobiology Second Edition
13Drawback is low efficiency of incorporation
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 14: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/14.jpg)
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
14
The bioorthogonal chemical reporter strategy for imaging glycans
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 15: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/15.jpg)
Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1
15
Imaging of Glycans in Living Zebrafish
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 16: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/16.jpg)
Proteomics 2006 6 5385ndash5408
16
Detection of Glycoproteins in SDS-PAGE
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 17: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/17.jpg)
Proteomics 2006 6 5385ndash5408
17
Detection of Glycoproteins in SDS-PAGE
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 18: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/18.jpg)
ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)
18
Detection of Glycoproteins in SDS-PAGE
periodic acid-Schiff (PAS) staining
Early work on RBC Surface Glycoproteins
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 19: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/19.jpg)
Clin Proteom (2009) 552ndash68
19
Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans
SYPRORuby total protein stain
Selective Staining of Glycoproteins on 2D-Gels
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 20: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/20.jpg)
Clin Proteom (2009) 552ndash6820
Lectin Blotting is a Powerful Tool to Detect Glycoproteins
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 21: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/21.jpg)
Clin Proteom (2009) 552ndash6821
DSL Lectin
LEL Lectin AAL Lectin
UEA I Lectin
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 22: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/22.jpg)
OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010
22
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 23: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/23.jpg)
O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23
Detection of Glycoproteins by Glycan-Specific Antibodies
Eg Pan-Specific antibody to O-GlcNAc
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 24: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/24.jpg)
24
Detection of Glycosphingolipids by Antibodies After TLC
Anal Chem 2009 81 9481ndash9492
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 25: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/25.jpg)
Anal Chem 2009 81 9481ndash9492
25
Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 26: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/26.jpg)
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
26
Typical Protocol Used to Analyze Glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 27: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/27.jpg)
Current Opinion in Chemical Biology 2007 1152ndash58
27
Affinity methods for isolation of glycoproteins
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 28: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/28.jpg)
Current Opinion in Chemical Biology 2007 1152ndash58
28
Unnatural sugars used in the isolation of glycoproteins
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 29: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/29.jpg)
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Options for Releasing Glycans From Proteins
29
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 30: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/30.jpg)
30
Endoglycosidases for N-Glycans
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 31: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/31.jpg)
31
PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally
(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 32: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/32.jpg)
Sigma-Aldrich Chemical Deglycosylation Strategies
32
Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically
PNGase F useful in Site Mapping by MS
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 33: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/33.jpg)
33
Sigma-Aldrich Glycoprotein Handbook
Unlike PNGase F O-Glycosidase is Highly Specfic
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 34: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/34.jpg)
Sigma-Aldrich Chemical Deglycosylation Strategies
34
Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 35: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/35.jpg)
Alkaline szlig-Elimination releases SerThr-linked Sugars
GlcNAc
O|
[3H]Gal[3H]Gal(1-4)GlcNAcitol
-Elimination
NaOHNaBH4
Often done with borohydride to prevent lsquopeelingrsquo35
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 36: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/36.jpg)
ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull
Differential glycan analysis using isotope labeling and mass spectrometry
36
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 37: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/37.jpg)
Alkaline induced-b Elimination
CNH
C
O
CH2
(Serine-O-GlcNAc)
(dehydroalanine)
CNH
C
O
CH2
OGlcNAc
H
Strategy for O-GlcNAcO-Phosphate site mapping
0
100
0
100
0
100
13143
PSVPVSerGSAPGR
O-GlcNAc
PSVPVSerGSAPGR
DTT
PSVPVSerGSAPGR
BAP
12474
14217
mz1100 1650
mz1100 1650
mz1100 1650
MALDI-TOF
Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition
CNH
C
O
CH2H
DTT (or BAP)
Michael Addition
HSCH2CHOHCHOHCH2SH (DTT)
1 Provides tag forAffinity enrichment
2 Tag is stable in massspectrometer
Relative intensity
Relative intensity
Relative intensity
BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)
37
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 38: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/38.jpg)
Mix
Performic acid treatment
lt--Trypsin--gt
O-GlcNAc-peptides O-phosphate-peptides
Control Cells Experimental Cells
Alkaline Phosphatase
BEMADHeavy DTT
LC-MSMS
Add Controls
BEMADLight DTT
O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase
LC-MSMS
Heavy and Light DTT labeled O-GlcNAc-peptides
Heavy and Light DTT labeled O-phosphate-peptides
Thiol Enrichment Thiol Enrichment
Johns Hopkins38
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 39: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/39.jpg)
Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites
BEMAD replaces the labile modification with a stable nucleoplilic group
J Am Chem Soc 125 (52) 16162 -16163 2003
39
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 40: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/40.jpg)
Sigma-Aldrich Chemical Deglycosylation Strategies
40
Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked
oligosaccharides ndashDestroys the Peptide
Anhydrous Hydrazine = Rocket Fuel
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 41: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/41.jpg)
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
Strategies for the analysis of released glycoprotein-glycans
41
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 42: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/42.jpg)
Bayer EA et al Meth Enzymol 184 415 (1990)42
Periodate Oxidation to Label Glycans
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 43: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/43.jpg)
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination
B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition
C labeling with phenylhydrazide and
D glycan permethylation
43
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 44: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/44.jpg)
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
44
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 45: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/45.jpg)
H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869
45
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 46: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/46.jpg)
46
Methods to Separate Released Glycans
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 47: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/47.jpg)
Tools of Glycome Profiling
47
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 48: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/48.jpg)
HPLC of Glycans
Carbohydrate Research 94 (1981) C7-CV
NormalPhaseChromatography
48
Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 49: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/49.jpg)
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
49
Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides
HPAEC
RP-HPLC
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 50: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/50.jpg)
Glycoprotein Analysis Manual Sigma-Aldrich
50
Normal Phase Chromatography of 2-AB labeled Glycans
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 51: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/51.jpg)
Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA
51
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 52: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/52.jpg)
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
52
Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B
Ovalbumin
Fetuin
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 53: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/53.jpg)
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
53
Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 54: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/54.jpg)
L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481
54
A natural glycan microarray approach with reductively aminated glycans
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 55: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/55.jpg)
55
Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids
Size After Desialylation
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 56: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/56.jpg)
Gel Filtration Chromatography ndash Separate Oligosaccharides by Size
56
Beta -Galase
Beta-Hexosaminidase
Alpha-Mannosidase
Beta-Mannosidase
Beta-Hexosaminidase
Alpha-Fucosidase
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 57: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/57.jpg)
Dionex with Pulsed-Amperometeric
Detection ndash A Breakthrough in the mid-80s
J Chromatogr A 720 (1996)295-321
At pH 13Carbohydratesionize and Become Charged molecules
57
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 58: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/58.jpg)
Derivatization of Glycans for
Electrophoresis
58
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 59: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/59.jpg)
Capillary Electrophoresis of Glycans
as published in BTi October 2004 59
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 60: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/60.jpg)
Fluorophore-Assisted Carbohydrate Electrophoresis
60
From Glyko
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 61: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/61.jpg)
Fluorophore-Assisted Carbohydrate Electrophoresis
(FACE)
61
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 62: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/62.jpg)
Hydrophilic Interaction Liquid Chromatography (HILIC)
62
(Formally called ldquoNormal Phaserdquo Chromatography)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 63: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/63.jpg)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
63
HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 64: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/64.jpg)
64
Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 65: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/65.jpg)
Chapter 47 Figure 2
Glycosidases used for structural analysis
Essentials of Glycobiology Second Edition
65
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 66: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/66.jpg)
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology
66
Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 67: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/67.jpg)
Chapter 47 Figure 3Essentials of Glycobiology
Second Edition
A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen
67
Methylation Analysis is Used to Determine Linkages
Enzymes or NMR are used to Determine Anomericity
Methyl Ether isAcid Stable
100 RXN
Reduction EliminatesAnomeric Mixtures
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 68: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/68.jpg)
Chapter 47 Figure 6
Data from a glycomics study of N-glycans from mouse kidney
Essentials of Glycobiology Second Edition
68
AssumptionsBased UponKnown Pathways
Typically Confirmed byMSMS and enzymes
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 69: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/69.jpg)
Current Opinion in Structural Biology 2009 19498ndash506
Example of MALDI-TOF Profiling of Glycans
69
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 70: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/70.jpg)
Chapter 47 Figure 4
1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Essentials of Glycobiology Second Edition
70
NMR is the mostPowerful MethodFor Glycan StructureDetermination
Provided 2 things are True
1 Enough Material
2 Oligosaccharide Is pure
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 71: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/71.jpg)
Chapter 47 Figure 5
NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F
Essentials of Glycobiology Second Edition
71
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 72: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/72.jpg)
Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure
Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society
Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation
Ion Trap MSA Powerful tool For Structural Determination of Glycans
72
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 73: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/73.jpg)
73
GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114
74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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74
Basic Structure of GPI-Anchors
75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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75
Strategy for Analysis of GPI-Anchors
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
![Page 76: -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153 Introduction to Glycan Analysis.](https://reader036.fdocuments.in/reader036/viewer/2022081513/56649e395503460f94b2a571/html5/thumbnails/76.jpg)
76
Chemical and enzymatic reactions of GPI anchors
Chapter 11 Figure 3Essentials of Glycobiology
Second Edition
77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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77
Characterization of Glycosaminoglycans
Bacterial EliminasesAre Powerful Tools
1 Sequential Degradation Followed by Gel Filtration
2 Other Separation Methods
78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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78
FACE Analysis of GAG-Derived Disaccharides
79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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79
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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80
Glycosyltransferases as Probes
81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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81
82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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82
83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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83
Galactosyltransferase Labeling of Living Cells O-GlcNAc
84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-
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84
Conclusions1 There are now a multitude of powerful methods to
analyze glycoconjugates
2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry
3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site
4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field
- -Techniques in Glycobiology-
- Techniques in Glycobiology
- Topics
- Slide 4
- Slide 5
- Slide 6
- Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed
- Compositional Analysis by HPAEC-PAD
- Compositional Analysis by GC-MS of Alditol Acetates
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Alkaline szlig-Elimination releases SerThr-linked Sugars
- Slide 36
- Slide 37
- Slide 38
- Slide 39
- Slide 40
- Slide 41
- Slide 42
- Slide 43
- Slide 44
- Slide 45
- Slide 46
- Slide 47
- HPLC of Glycans
- Slide 49
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex
- Gel Filtration Chromatography ndash Separate Oligosaccharides by Si
- Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in
- Derivatization of Glycans for Electrophoresis
- Capillary Electrophoresis of Glycans
- Fluorophore-Assisted Carbohydrate Electrophoresis
- Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
- Hydrophilic Interaction Liquid Chromatography (HILIC)
- Slide 63
- Slide 64
- Slide 65
- Slide 66
- Slide 67
- Slide 68
- Slide 69
- Slide 70
- Slide 71
- Slide 72
- Slide 73
- Slide 74
- Slide 75
- Slide 76
- Slide 77
- Slide 78
- Slide 79
- Slide 80
- Slide 81
- Slide 82
- Slide 83
- Slide 84
-