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INTRODUCTION TO ANALYTICAL GLYCOBIOLOGY
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Transcript of INTRODUCTION TO ANALYTICAL GLYCOBIOLOGY
GLYCOMICS Workshop17th IMSC, Prague, 2006
Milos V. Novotny, Ph.D.Yehia Mechref, Ph.D.
www.ncgg.indiana.eduFunded by NIH-NCRR
GLYCOBIOLOGYGLYCOPROTEOMICSFUNCTIONAL GLYCOMICS
Introduction
IntroductionProteomics
Klose, J. Electrophoresis (1999) 20, 643-652.
IntroductionThe Polygenic Nature of Proteins
PROTEIN PHENESPROTEIN PHENES
Primary structureCleavage productsPolymerismsSide chainsConformationsBinding propertiesSynthesis ratesDegradation ratesTissue Specific amountsStage specific amounts
Structural gene
Regulatory genes transcription factors
Regulatory genesregulatory proteins
Regulatory sequences(enhancer acting in trans)
Regulatory sequences(locus control regions)
Genes involved in chromatin structures
Modifier genes Position effects Genes involved inof genes DNA methylation
Genes enzymes catalyzingposttrancriptionalmodifications of proteins
Genes enzyme catalyzing protein processing
Genes enzyme catalyzing transport and integration of proteins
Genes enzyme catalyzing protein degradation
PROTEIN PHENESPROTEIN PHENES
Primary structureCleavage productsPolymerismsSide chainsConformationsBinding propertiesSynthesis ratesDegradation ratesTissue Specific amountsStage specific amounts
Structural gene
Regulatory genes transcription factors
Regulatory genesregulatory proteins
Regulatory sequences(enhancer acting in trans)
Regulatory sequences(locus control regions)
Genes involved in chromatin structures
Modifier genes Position effects Genes involved inof genes DNA methylation
Genes enzymes catalyzingposttrancriptionalmodifications of proteins
Genes enzyme catalyzing protein processing
Genes enzyme catalyzing transport and integration of proteins
Genes enzyme catalyzing protein degradation
Introduction
23 MARCH 2001"Carbohydrates and Glycobiology"Vol. 291, Pages 2263-2502
The cell surface landscape is richly decorated with oligosaccharides anchored to proteins or lipids within the plasma membrane. Cell surface oligosaccharides mediate the interactions of cells with each other and with extracellular matrix components.
The important roles that carbohydrates play in biology and medicine have stimulated a rapid expansion of the field of glycobiology
Introduction
IntroductionGlycosylation
The most common post-translational modification of proteins: membrane bound receptors, many soluble proteins and even nuclear proteins.
Oligosaccharides substitution occurring at asparagine residues (N-linked) and threonine or serine residues (O-linked)
Microheterogeneities: statistical or on purpose?
IntroductionSome Functions of Oligosaccharides
Recognition markers(“recognition markers” can be put on quite different proteins without coding the information into DNA)Increased stability and protease resistanceFolding and maintaining the advanced structures
GLYCOSYLATION is protein-specific, site- specific and tissue/cell-specific
MASS SPECTROMETRY is the key methodology to address protein and glycoconjugate sequencing, differential quantitative measurements, extent of protein modifications and, to some degree, biomolecular complexation phenomena. We utilize different ionization techniques (ESI, MALDI), different mass separation technologies (ion trap, TOF, ICR, IMS, etc.) and their tandem modes, and different detection techniques to accomplish these tasks.MS measurements are greatly assisted by proper combinations (on- or off-line) with modern SEPARATION METHODS, including affinity chromatography, 2-D electrophoresis, capillary LC and electrophoretic methods (CEC and CZE). They provide prefractionation and/or discrete separation of the complex mixtures of biological molecules, enhancing the SENSITIVITY of final measurements Extensive measurements and complex analytical data are interpreted through the extensive use of BIOINFORMATICS in both proteomic and glycomic investigationsMICROCHEMICAL PROCEDURES (e.g., sample derivatizations or enzymatic treatments) are significant in the overall success of glycomic and glycoproteomic measurements
Introduction
IntroductionA Brief History of Protein Sequencing
Per Edman, the 1950s and 60s: chemical approach and chromatography.Klaus Biemann, since the 1960s: mass spectrometry of derivatized, small peptides (GC/MS, EI and CI)Introduction of FAB during the early 1980s extends the scopeInventions of ESI and MALDI dramatically change our capabilities during the late 1980s.Gradual evolution of microseparationtechniques, new MS capabilities and bioinformatics continues to this date.
Introduction
Sequencing of Peptides
y1
y2
y3
y4
y5
y7
y8 y9
y10 y11 y13
Mass (m/z)
% In
tens
ity
1.7E+4
K +42 S F T S T S D W N D L L +184 +42y6
69.0 443.4 817.8 1192.2 1566.6 1941.00
10
20
30
40
50
60
70
80
90
100
712.
32
276.
14
611.
27
423.
20
189.
12
898.
42
1013
.44
524.
24
811.
40
1199
.53
1313
.55
1428
.60
1541
.71
y12
1838
.70
Precursor ion
V
IntroductionProteomics Approaches
Bottom-up Top-down
Lysecells
Digest proteins
Separate proteins
Protein MS
Protein MS/MS
Peptide MS &
MS/MS
Digest proteins
Separate peptides
LC2D-LC
Peptide MS &
MS/MS
Peptide MS &
MS/MS
Advantages
LC resolution
MS sensitivity
Sequence Coverage
PTM ID
Bottom-up Top-down
Lysecells
Digest proteins
Separate proteins
Protein MS
Protein MS/MS
Peptide MS &
MS/MS
Digest proteins
Separate peptides
LC2D-LC
Peptide MS &
MS/MS
Peptide MS &
MS/MS
Advantages
LC resolution
MS sensitivity
Sequence Coverage
PTM ID
Introduction2-D gel images of Extracts from Liver Tissues of Alcohol Naïve and Alcohol Exposed Rats.
3304
3313
3520 3545
4525
4520
3343
3340
3253
4332
44405604
5607
4136
5126
5512
55185533
65066514
5421 5436
6408
6312
6432
6438
53335315
5325
5328
5337
6320
63307413
7311
63327303
7210
7329
82028201
713272097107 7118
61126111
6205
6213
5235
6238
6212
4506
pI3 10
200.0
97.4116.25
66.2
45.0
31.0
21.5
14.4
6.5
kDa
3304
3313
3520 3545
4525
4520
3343
3340
3253
4332
44405604
5607
4136
5126
5512
55185533
65066514
5421 5436
6408
6312
6432
6438
53335315
5325
5328
5337
6320
63307413
7311
63327303
7210
7329
82028201
713272097107 7118
61126111
6205
6213
5235
6238
6212
4506
pI3 10
200.0
97.4116.25
66.2
45.0
31.0
21.5
14.4
6.5
kDa
116.25
66.2
45.0
31.0
21.5
14.4
6.5
kDa200.097.4
pI
3304
3313
3520 3545
4525
4520
3343
3340
3253
4332
44405604
5607
4136
5126
5512
55185533
65066514
5421 5436
6408
6312
6432
6438
53335315
5325
5328
5337
6320
6330
7311
7210
7329
82028201
713272097107 7118
61126111
6205
6213
5235
6238
6212
4506
7413
63327310
3 10
116.25
66.2
45.0
31.0
21.5
14.4
6.5
kDa200.097.4
pI
3304
3313
3520 3545
4525
4520
3343
3340
3253
4332
44405604
5607
4136
5126
5512
55185533
65066514
5421 5436
6408
6312
6432
6438
53335315
5325
5328
5337
6320
6330
7311
7210
7329
82028201
713272097107 7118
61126111
6205
6213
5235
6238
6212
4506
7413
63327310
3 10
I. Klouckova, P. Hrncirova, Y. Mechref, R. J. Arnold, T.-K. Li, W. J. McBride, M. V. Novotny “Changes in liver protein abundance in inbred alcohol-preferring rats due to chronic alcohol exposure measured through a proteomics approach” Proteomics, 2006, 6, 3060-3074.
O O
C CO O
O-GlycanO-Glycan
O-Glycan
O-Glycan
GPI MembraneAnchor
N-Glycan
N-AcetylglucosamineN-AcetylgalactosamineMannoseGalactoseFucoseSialic acidGlucosamineInositolEthanolaminePhosphate
Introduction
CARBOHYDRATES: The Most Abundant Class of Biological Molecules
In various polysaccharides (cellulose, starch, pectin, chitin, glycogen, hyaluronic acid, etc.), aiding architecture of living cells and serving in energy storage and metabolismAs oligosaccharides in association with proteins (glycoproteins)and lipids (glycolipids), serving various biological function (recognition determinants)As glycosaminoglycans, the highly charged, unbranchedpolysaccharides of alternating uronic acid and hexosamineresidues, which are the essential components of proteoglycans, the giant molecules of glycobiologyAs the essential components of a rapidly growing number of biocompatible materials derived from the chemical modifications of polysaccharides, and a number of biotechnologically-derived and synthetic compounds
IntroductionBASIC TERMS AND NOMENCLATURE
Monosaccharides (basic units) oligosaccharides (linear or branched structures) polysaccharidesGlycans and glycoconjugatesAnomericity and different linkages between the monosaccharide unitsStereochemical relationships (D-stereochemistry preserved from D-glyceraldehyde through hexosesugars)Different branched structures (e.g., biantennary, triantennary, tetra-antennary glycans)Different core structures (consensus sequences) for differently linked glycans
The anomeric monosaccharides α-D-glucopyranose and β-D-glucopyranose, drawn as both Haworth projections and ball-and-stick models. These pyranosesugars interconvert through the linear form of D-glucose and differ only by the configuration about their anomeric carbon atoms, C(1).
IntroductionBASIC TERMS AND NOMENCLATURE
IntroductionBasic Terms and Nomenclature
Introduction Monosaccharides Commonly Found in Glycoproteins
(CH2OH)
N-glycolylneuraminic acid (NeuGc)
IntroductionGlycan Heterogeneity
A linear oligomer of DNA consisting of 3 monomers could have a maximum of 64 isomers.A protein of the same length could have 8000 isomers.An oligosaccharide could have 64 000isomers.
IntroductionGlycan Heterogeneity
IntroductionThree Types of N-Linked Glycans
Manα1-2Manα1\ 6
Manα1-2Manα1-3Manα1\ 6Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn/ 3
Manα1-2Manα1-2Manα1
Neu5Acα2-3Galβ1-4GlcNAcβ1\ 4
Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1\ 6
Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn/ 3
Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1/ 4
Neu5Acα2-3Galβ1-4GlcNAcβ1/ 3
Fucα1
triditetra
Complex type
Manα1\ 6
Manα1-3Manα1\ 6
GlcNAcβ1-4 Manβ1-4GlcNAcβ1-GlcNAcβ1-Asn/ 3
Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1
High-mannose type
Hybrid type
tri’
IntroductionCore Structures of Mucin Type O-Linked Glycans
Type
Core 1
Core 2
Core 3
Structure Type Structure
Galβ1-3GalNAc
GlcNAcβ1-6
Galβ1-3GalNAc
GlcNAcβ1-3GalNAc
Core 4
Core 5
Core 6
GlcNAcβ1-6
GlcNAcβ1-3GalNAc
GalNAcα1-3GalNAc
GlcNAcβ1-6
GalNAc
GalNAcα-Ser (Thr)
Glycan heterogeneity in the N-glycosylation sites of plasma and cellular fibronectins (2350 residues and seven N-glycosylation sites)
Y. Wada et al.
IntroductionStructural Information Needed
Site of glycosylationSite occupancySequencesDefinition of branchingLinkages and configurationDistinction of isobaric structures
Fragmentation of glycans observed in MALDI/MS is similar to that observed in FAB/MS and ESI/MS and is dependent on factors such as
ion formation, its charge state, the energy deposited into an ion, and the time available for fragmentation.
Fragmentation of Glycans
Fragmentations in MALDI/MS can result from
(a) the post-source decay (PSD) which designates the fragments formed after ion extraction from the ion source, (b) in-source-decay (ISD) which designates the fragments formed within the ion source, and (c) collision-induced dissociation (CID) which designates the fragments formed in a collision cell filled with a gas.
Fragmentation of Glycans in MALDI-MS
B1 C1 B2 C2 B3 C3
Z0Y0Z1Y1Z2Y2
A. Glycosidic FragmentationA. Glycosidic Fragmentation
OOH
OH
CH2OH
O
CH2OH
OH
OH
OHO
OOH
OH
CH2OH
O O R
Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.
Glycomic AnalysisFragmentation of Glycans in MALDI-MS
Glycomic AnalysisFragmentation of Glycans in MALDI-MS
Y. Mechref, A. G. Baker, M. V. Novotny, Carbohydrate Res., 313 (1998): 145–155.
B. CrossB. Cross--ring Fragmentationring Fragmentation
0,2A1
1,5X13,5X2
2,4A22,5A3
OOH
OH
CH2OH
O
CH2OH
OH
OH
OHO
OOH
OH
CH2OH
O O R
Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.
Glycomic AnalysisFragmentation of Glycans in MALDI-MS
Glycomic AnalysisFragmentation of Glycans in MALDI-MS
9.0 271.6 534.2 796.8 1059.4 1322.0Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
22.9
1036
.4
1257
.552
4
1123
.5
671.
2
849.
3
799.
3
475.
2
1095
.4
907.
3265
447.
2
73.3
226.
1
41.1
653.
2
164.
9 347.
1
527.
2
244.
1 1061
.4
771.
3
874.
3
509.
255
0.2
979.
3621
429.
2
583.
2
272.
1
185.
1
625.
2
399.
2
689.
2
147.
8
327.
230
6.9
1156
.467
3
745.
3
953.
3788
203.
1
817.
3
365.
1
712.
2
1010
.396
8
91.4
1182
.481
6
933.
3752
607.
2
1,5 X
4x,3β
0,2 A
53,
5 X4x
,3β
B4
C4
1,5
1,5 XX
33 αα
Y2
C3
C2α
B2α
Ion DB3/Y3β
B3
Y3α
Y4x
2,4 A
5
0,2 A
4
3,5 A
4
3,5
3,5 AA
33
0,4
0,4 AA
33
Z2
1,5 X
2
(Hex)2
1,5 X
1
Y1
Z1
B 4/Y
4x,3β
3,5X4α’
0,40,4AA33
3,53,5AA33
3,5 A
5
3,5A53,5X4α”
3,5X3β
1,51,5XX33αα
1,4A5
0,2A5
1,5X4α’
1,5X4α”
1,5X3β
0,2A4
3,5A4
1,51,5XX22
2,4A5
Na+
B1
C1
1,5 X
0
655.
2B 3
/Z 3
β
OCH2OH
OH OOH
OH
O
CH2OH
OH O
O
CH2OH
OH
NH
OH
CCH3
O
O
O
CH2OH
OH
NH
CCH3
O
O
O
O
CH2
OCH2OH
OH
OH
OH
OH
OH
OH
OO
CH2OH
OH OH
OH
Yehia Mechref, Milos V. Novotny and Cheni Kirshnan, Anal. Chem., Anal. Chem., 75 (2003), 4895-4903.
Glycoconjugate BiosynthesisGlycoconjugate Biosynthesis
Carolyn R. Bertozzi and Laura L. Kiessling, Science, 291 (2001) 2357-2364
Some Glycoconjugate Disease Associations
immunity to infectious diseases, including HIVrheumatoid arthritis (altered composition of IgG and levels of the serum mannose-binding protein)prion diseasescongenital disorders of glycosylation (rare, usually resulting in CNS impairment)oral pathologiescystic fibrosisheart pathologiescancer
Congenital Disorders of Glycosylation (CDG) As Example
Related to errors in fundamental glycosylation machinery; also, relevant to various aspects of developmental biology where biosynthesis, monosaccharide additions or processing may differMore than 20 CDGs now identified (most, but not all, involve N-glycans)In some CDGs, multiple pathways seem involved; disruption of traffic in Golgi glycosylation machineryAt their recent meeting in Osaka, Japan, Human Disease Glycomics/Proteome Initiative (HGPI) directors have suggested that CDGs be employed as a model for unifying methodologies in functional glycomics
Jaeken and Matthijs, Annu. Rev. Genomics Hum. Genet. 2: 129-51 (2001)
Congenital Disorders of Glycosylation (CDG) As Example
A. Dell and H. Morris, Science 291, 2001
Congenital Disorders of Glycosylation (CDG) As Example
Increased branching and sialylation of N-linked glycans; fucosylationIncreased sialylation of O-linked glycansOccurrence of polysialic acidTruncation of some O-linked structures
Most current emphasis on O-linked structures = methodological challenges
Glycosylation Pertaining To Cancer: Representative Changes
L. Tong, et al. in Biotechnology & Genetic Engineering Reviews, Vol 20, p. 214 (2003)
Glycosylation Pertaining To Cancer: Representative Changes
Glycomic AnalysisPlatform
Human Blood Serum
GLYCOMIC MAPS
Protein Extraction
Glycopeptides
Glycoproteins
N-GLYCANS
MALDI TOF / MS
N-Glycans
Reduction & Alkylationwith TFE
Tryptic Digestion
PNGase F Digestion
Solid Phase Extraction
Permethylation
Human Blood Serum
GLYCOMIC MAPS
Protein Extraction
Glycopeptides
Glycoproteins
N-GLYCANS
MALDI TOF / MS
N-Glycans
Reduction & Alkylationwith TFE
Tryptic Digestion
PNGase F Digestion
Solid Phase Extraction
Permethylation
1500 2200 2900 3600 4300 5000Mass (m/z)
Healthy
Breast Cancer Stage IV
Glycomic AnalysisPlatform
0
20
40
60
80
100
high mannose hybr id complex-BI complex-BI-Fuc complex-Tr i complex-Tr i -Fuc complex-Tetr a complex-Tetr a-Fuc Fucosylated Sialylated
NORMAL STAGE IV
0
10
20
high m
anno
se
hybri
d
comple
x-BI
comple
x-BI-F
uc
comple
x-Tri
comple
x-Tri-
Fuc
comple
x-Tet
raco
mplex-T
etra-
Fuc
Fuco
sylat
ed
Sialy
lated
%re
l. inte
nsity
NORMAL STAGE IV
Comparing Glycomic Profiles of Normal (n=27) vs. Stage IV of Breast Cancer (n=50)
Glycomic AnalysisFunctional Glycomics
Isolation of Glycoproteins
Resolution of isoforms in gels and CEIsolation schemesUse of lectinsExamples of isolation/structural determinations
Analysis of GlycoproteinsCapillary Electrophoresis
Effect of additives on the CZE separation of rHuEPO: Sample: 1 mg/ml; fused-silica capillary (50 cm_75 mm i.d.); voltage: 10 kV. Buffers at pH 6.2: (A) 10 mMtricine/10 mM NaCl; (B) 10 mMtricine/10 mM NaCl/2.5 mM 1,4-diaminobutane; (C) 10 mMtricine/10 mM NaCl/2.5 mM 1,4-diaminobutane/7 M urea. UV detection at 214 nm.
E. Watson, F. Yao, J. Chromatogr. 630 (1993) 442.
Isolation of GlycoproteinsGeneral Considerations
Glycoproteins are often encountered in minute quantities in biological materials such as cellular extracts and physiological fluidsThere is high demands placed on both the measurement sensitivity and proper isolation procedures. A combination of orthogonal separation techniques and the use of affinity principles are the most commonly practiced isolation/fractionation strategies.Miniaturization of these separation and isolation/fractionation methodologies represents a general trend in glycoanalysis.
Glycoproteins at the low-microgram scale, while becoming measurable with the modern instrumental techniques, can easily be adsorbed on the surface of glassware before such measurements. Sample loss during ultrafiltration, dialysis, lyophilization, etc., can easily become a bottleneck of the entire analysis. Another problem with working at such a reduced scale is contamination (dust, solvent, reagent impurities, etc.). It is thus crucial to minimize the number of handling and transfer steps during the analysis. Miniaturized forms of separation, in terms of
reduced column diameters, solvent flow-rates and the overall surface area that a glycoprotein sample may encounter during analysis, are becoming significant in high-sensitivity work.
Isolation of GlycoproteinsGeneral Considerations
Glycoproteins can be purified by most conventional protein separation methodologies, including
gel electrophoresis various forms of HPLC (ion-exchange, size exclusion, reversed phase using C18, C8 or C4 columns, hydrophobic interaction, and affinity).
A most useful, specific isolation principle is the use of lectins that are immobilized on chromatographic resins
Isolation of GlycoproteinsGeneral Considerations
Y. Mechref and M.V. Novotny, Chem. Rev., 102, 321, (2002).
2-D Gel Electrophoresis
Biological SampleBiological Sample
Affinity Chromatography
GlycoproteinsGlycoproteins
SDS-PAGEReversed-PhaseChromatography
Ion-Exchange Chromatography
Isolated GlycoproteinsIsolated Glycoproteins MALDI/TOF MS
Proteolytic Enzyme Digestion
HPCE-MS μLC-MS
“Fingerprint” of the Glycoprotein“Fingerprint” of the Glycoprotein
MS/MS Database Search
Elucidation of Amino Acid Sequence and Glycosylation Sites
Elucidation of Elucidation of Amino Acid Sequence and Glycosylation SitesAmino Acid Sequence and Glycosylation Sites
MALDI/TOF MS
Mixture ofPeptides and Glycopeptides
Mixture ofPeptides and Glycopeptides
Isolation of GlycoproteinsStrategies for Analysis
Analysis of Glycoproteins2-DE
In 2-DE separations, glycoproteins tend to be translocated into “trains” of spots, reflecting their differences in both the molecular mass and isoelectric points.Silver-stained serum/plasma proteins after 2-DE.
(1) transferrin, (2) IgM m chain, (3) IgA a chain, (4) α1-antitrypsin, (5) haptoglobin b chain and haptoglobin cleaved b chain, (6) Ig light chains, (7) IgG g chain.
Gravel, P.; Walzer, C.; Aubry, C.; Balant, L. P.; Yersin, B.; Hochstrasser, D. F.; Guimon, J., Biochem. Biophys. Res. Commun. 1996,220, 78-85.
The Challenge of Protein Identification in Human Sera
Number of Proteins
MS
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
Immunoaffinity +MS
Con
cent
ratio
n (g
/ml)
Current tissue biomarker research activities
Proteins of Interest
Lectins have in the past been regarded by many scientists as curious proteins of uncertain structure and specificity that bind to carbohydrates of dubious significance themselves. All this is rapidly changing. The functional importance of glycosylation in cell-cell and cell-pathogen interactions, as well as intracellular events, has been recognized by the explosion of the science of glycobiology. This has been paralleled by the realization that lectins, once they have been well characterized, can be extremely useful tools for examining structural changes in glycosylation and their functional consequences for human pathophysiology.
Preface to “Lectin Methods and Protocols,”by Jonathan M. Rhodes and Jeremy D. Milton, Humana Press, 1998
Isolation of GlycoproteinsLectin Affinity Chromatography
Lectins are specialized proteins that have been isolated from various plants and animal sources. Lectins have been widely used to isolate, purify and characterize glycoproteins and glycolipids in various modes of affinity chromatography. These techniques are based on a reversible biospecific interaction of certain glycoproteins with the lectins immobilized to a solid support.
Isolation of GlycoproteinsLectin Affinity Chromatography
Isolation of GlycoproteinsLectin Affinity Chromatography
Table 1. Commonly used agarose-based lectins.
Name or abbreviation Source Amount of
immobilized lectin [mg/mL]
Specificity
Con A (Concanavalin A) Canavalia ensiformis 8 - 10 Glc, Man SNA-I Sambucus nigra 2 - 3 NeuAc α(2,6) MAA Maackia amurensis 2 -3 NeuAc α(2,3) UEA-I Ulex europaeus 4 - 5 Fuc α(1,2)
Jacalin Artocarpus integrifolia 2 - 3 Gal
PHA-L Phaseolus vulgaris 4 - 5 complex Lotus Lotus tetragonolobus 4 -5 Fuc α(1,2) HPA Helix pomatia 1 -2 GalNAc WGA (Wheat germ) Triticum vulgaris 4 -5 GlcNAc RCA-I (Ricin) Ricinus communis 4 -5 Gal, GalNAc LcH (Lentil) Lens culinaris 4 - 5 Man EY Laboratories (St. Mateo, CA)
GlcNAcGalMan
NeuAcFuc
Con A
SNA or MALLotus Lectin
SNA or MAL
Lentil LectinPEA LectinSNA or MAL
LectinsCon A – Canavalia ensiformis SNA – Sambucus nigra MAL – Maackia amurensisLotus Lectin – Tetragonolobus purpureas PEA Lectin – Phaseolus vulgaris Lentil Lectin – Lens
culinaris
R.D.Cummings, Methods in Enzymology, Vol. 230 (1994) 66-86
Isolation of GlycoproteinsLectin Specificity
Isolation of GlycoproteinsLectin Affinity Chromatography
Equilibrating with binding buffer
Applying sample prepared in binding buffer, prompting specific, but reversible, binding of target substances, while other material will wash through
Elution of bound material, using a competitive ligand which displaces target substances
Re-equilibrating with binding buffer
0 5 10 15 20 25 30
0204060
mA
U
Time [min]
0 5 10 15 20 25 30
0204060
mA
U
Time [min]
0 5 10 15 20 25 30
0204060
mA
U
Time [min]
0 5 10 15 20 25 30
0204060
mA
U
Time [min]
Equilibrating with binding buffer
Applying sample prepared in binding buffer, prompting specific, but reversible, binding of target substances, while other material will wash through
Elution of bound material, using a competitive ligand which displaces target substances
Re-equilibrating with binding buffer
0 5 10 15 20 25 30
0204060
mA
U
Time [min]
0 5 10 15 20 25 30
0204060
mA
U
Time [min]
0 5 10 15 20 25 30
0204060
mA
U
Time [min]
0 5 10 15 20 25 30
0204060
mA
U
Time [min]
Y. Mechref, L. Zidek, W. Ma, and M.V. Novotny, Glycobiology, 10 (2000) 231-235.
*
*
Anion-exchange chromatogram of the isolated MUP components (upper trace) and the Concanavalin A bound fraction (lower trace); inset is the mass spectrum of the isolated glycoprotein, as indicated with the asterisk.
Isolation of GlycoproteinsLectin Affinity Chromatography
Y. Mechref, L. Zidek, W. Ma, and M.V. Novotny, Glycobiology, 10 (2000) 231-235.
934.
477
1138
.52
1299
.92
1342
.02
1503
.08
1665
.55
1706
.78
1340
.52
1502
.55
1544
.17
1664
.58
1705
.79
1749
.58
1841
.92
1868
.05
1908
.31
1663
.85
2029
.07
2395
.03
-40000
-30000
-20000
-10000
0
10000
20000
Cou
nts
1000 1500 2000 2500 3000 Mass (m/z)
andand
andand
andand
Isolation of GlycoproteinsLectin Affinity Chromatography
SDS-PAGE, Water Soluble Glycoproteins in the VNO of MiceMW
markerMW
markerImmature
femaleAdultfemale
Immaturemale
Adult male
70 kD
22 kD
14.4 kD
21.5 kD
45.0 kD
66.2 kD
97.4 kD
200 kD
VesomeralsecretoryProtein I(VNSP I)
SerotransferrinPrecursor
Analysis of GlycoproteinsSDS-PAGE
Glycosylation Site of VNSP I
20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00Time0
100
%
0
100
%
0
64.6
5
51.9
8
65.0
4
70.4
3
44.7
040
.40
37.1
0
34.8
733
.93
30.1
8
42.8
1 59.4
2
46.7
1
57.0
5
52.3
351
.57
50.0
2 68.8
0
62.4
5
63.9
6
79.8
6
73.2
375
.34 99
.50
97.0
295
.82
82.3
9
88.6
3
100.
2010
2.75
250 750 1250 1750 2250 2750 3250 3750 4250 4750 5250mass0
100
%
x42 3755
204.
0737
3610
.392
3
366.
1324 528.
1738
3406
.075
7
569.
2050
1563
.462
9
782.
3042
1053
.929
8
1805
.758
4
2516
.134
3
2142
.980
7
3004
.488
828
74.4
055
4854
4708
.778
3
4446
.612
841
37.7
490
3959
.233
2
5059
.231
0
Product Ion Chromatogram
MSMS Spectrum of Glycopeptide
TIC Chromatogram
Glycosylation site [LAFNNGNFSGK]
20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00Time0
100
%
0
100
%
0
64.6
5
51.9
8
65.0
4
70.4
3
44.7
040
.40
37.1
0
34.8
733
.93
30.1
8
42.8
1 59.4
2
46.7
1
57.0
5
52.3
351
.57
50.0
2 68.8
0
62.4
5
63.9
6
79.8
6
73.2
375
.34 99
.50
97.0
295
.82
82.3
9
88.6
3
100.
2010
2.75
250 750 1250 1750 2250 2750 3250 3750 4250 4750 5250mass0
100
%
x42 3755
204.
0737
3610
.392
3
366.
1324 528.
1738
3406
.075
7
569.
2050
1563
.462
9
782.
3042
1053
.929
8
1805
.758
4
2516
.134
3
2142
.980
7
3004
.488
828
74.4
055
4854
4708
.778
3
4446
.612
841
37.7
490
3959
.233
2
5059
.231
0
Product Ion Chromatogram
MSMS Spectrum of Glycopeptide
TIC Chromatogram
Glycosylation site [LAFNNGNFSGK]
Analysis of GlycoproteinsIn-gel Digestion and LC/MSMS Analysis
Analysis of GlycoproteinsMultidimensional Approach
Sample
MARS Column
On-line microcolumn lectinaffinity chromatographywith high-temp RPHPLC
Collection ofthe fractions
Tryptic Digestion
Nano LC-MS/MS Database search Result
Lectinmicrocolumn
Isocratic pumpwith the injector
Capillarypump
Waste
C4 trappingcolumn
PoroshellC8 microcolumn
Lectinmicrocolumn
Isocratic pumpwith the injector
Capillarypump
Waste
C4 trappingcolumn
PoroshellC8 microcolumn
Sample
MARS Column
On-line microcolumn lectinaffinity chromatographywith high-temp RPHPLC
Collection ofthe fractions
Tryptic Digestion
Nano LC-MS/MS Database search Result
Lectinmicrocolumn
Isocratic pumpwith the injector
Capillarypump
Waste
C4 trappingcolumn
PoroshellC8 microcolumn
Lectinmicrocolumn
Isocratic pumpwith the injector
Capillarypump
Waste
C4 trappingcolumn
PoroshellC8 microcolumn
M. Madera. Y. Mechref, I. Klouckova, M.V. Novotny, J. Proteome Res., 2006; 5(9); 2348-2363.
Silica-Based Con A
Silica Resins FITC-Con A Immobilized
Texas RedOvalbuminBound to FITC-Con A
Milan Madera, Yehia Mechref and Milos V. Novotny “Combining Lectin Microcolumns With High-Resolution Separation Techniques For Enrichment Of Glycoproteins And Glycopeptides”, Anal. Chem., 77(13) (2005) 4081-4090.
Analysis of GlycoproteinsMultidimensional Approach
0 10 20 30 40 50 60
0
200
400
600
800
1000
1200
1400
Abso
rban
ce a
t 214
nm
Time [min]0 10 20 30 40 50 60
0
200
400
600
800
1000
1200
1400
Abso
rban
ce a
t 214
nm
Time [min]
0
510
15
20
2530
35
40
4550
55
60
Proteins
M. Madera. Y. Mechref, I. Klouckova, M.V. Novotny, J. Proteome Res., 2006; 5(9); 2348-2363.
Analysis of GlycoproteinsMultidimensional Approach
0
200
400
600
800
1000
PHA
- L
UEA
SNA
MW
[kD
a]
Con
A
0
50
100
150
200
250
300
350
PHA
-L
UEA
SNA
MW
[kD
a]
Con
A0
200
400
600
800
1000
PHA
- L
UEA
SNA
MW
[kD
a]
Con
A
0
50
100
150
200
250
300
350
PHA
-L
UEA
SNA
MW
[kD
a]
Con
A
M. Madera. Y. Mechref, I. Klouckova, M.V. Novotny, J. Proteome Res., 2006; 5(9); 2348-2363.
Analysis of GlycoproteinsMultidimensional Approach
2006; 5(9); 2348-2363.
Glycoproteome Changes in the Blood Serum of Healthy and Breast Cancer Patient
No.
of P
eptid
es
0
2
4
6
8
10
12
14
16G
PV_H
UM
AN
HB
D_H
UM
AN
TGM
3_H
UM
AN
TRFE
_HU
MA
N
CPG
L2_H
UM
AN
DO
PO_H
UM
AN
HO
RN
_HU
MA
N
CPG
L2_H
UM
AN
SCC
A2_
HU
MA
N
HPT
R_H
UM
AN
CancerHealthy
Con A Lectin Enrichment
Analysis of GlycopeptidesGeneral Considerations
It is most common that glycosylation of a particular protein is investigated through a chemical or enzymatic release of glycans and their subsequent characterization such as sequencing and linkage analysis.
While this information may be highly significant, there are additional structural aspects that must be addressed.
For each site of glycosylation, there are possible structural variations (extent of substitution, site-specific microheterogeneities, a site-specific accessibility for particular glycosyltransferases, etc.), which can all have important biochemical consequences.
Investigating protein glycosylation at the level of glycopeptide is at least as important as the investigation of released glycan structures.
It is difficult to directly identify glycopeptides in a complex protein digest by MS.
This is partly due to the low sensitivity of the detection of glycopeptides caused by
site heterogeneity and/or ion adduct formation. Glycopeptide signals are often suppressed in the presence of other peptides, especially if the glycans are terminated with the negatively charged sialic acid moiety.
Due to the glycan heterogeneity and a frequent multiple adduct formation, the overall glycopeptidesignal distributes into several peaks resulting in weak signals detected by MS.
Analysis of GlycopeptidesGeneral Considerations
Analysis of GlycopeptidesMALDI/MS (Partial 18O-lableing)
The inset in the bottom panel shows an expanded view of the molecular ion region for one of the formerly glycosylated peptides. Due to the small number of sequence-related peptide ions in the top spectrum, protein identification was ambiguous. The increased number of peptides obtained after deglycosylation together with the NXS/T sequence pattern allowed identification with certainty.
Sequence-specific peptide ion signals are marked with bullets, matrix-related peaks are labeled "M", and trypsin autolysis products are denoted with an asterisk.
Kuster, B.; Mann, M. Anal.Chem. 1999, 71, 1431-1440.
MALDI HIVrgp120 (in-gel trypsin digestion)
MALDI HIVrgp120 (in-gel PNGase F andtrypsin digestion)50% 18O-labeled water
Analysis of GlycopeptidesMALDI/MS (Alkylaminylation)
Alkylaminylation and clostripain digestion of human MUC1 from milk fat membranes.
Clostripain cleaves the MUC1repeat domain into icosapeptides of the sequence PAPGSTAPPAHGVTSAPDTR (calculated monoisotopic mass of MH 1886.9). (A) Ethylaminylated products were identified via their average masses to be substituted with one (m/z 1915.3), two (m/z 1941.3), three (m/z 1968.4), and four EA residues (m/z 1995.5); (B) Methylaminylated products were identified via their average masses to be substituted with no (m/z 1887.4), one (m/z1899.4), two (m/z 1912.4), three (m/z1925.5), four (m/z 1938.4), and five MA residues (m/z 1951.5).
Hanisch, F. G.; Jovanovic, M.; Peter-Katalinic, J. Anal. Biochem. 2001, 290, 47-59.
Analysis of GlycopeptidesMALDI/MS (Pronase Digestion)
An, H. J.; Peavy, T. R.; Hedrick, J. L.; Lebrilla, C. B. Anal. Chem.; 2003; 75(20); 5628-5637.
(a) MALDI-FT MS spectrum from Pronasedigestion of X. laeviscortical granule lectin (XL CGL). The most abundant peak, m/z 2168, is a self-proteolysis product of Pronase.
High-mannose-type glycans were labeled by and and hybrid/complex type glycans were labeled by and O at 154Asn and 217Asn, respectively.
Analysis of GlycopeptidesMALDI/MS (Pronase Digestion)
An, H. J.; Peavy, T. R.; Hedrick, J. L.; Lebrilla, C. B. Anal. Chem.; 2003; 75(20); 5628-5637.
Analysis of GlycopeptidesESI/MS
Y. Mechref, J. Muzikar, M.V. Novotny, Electrophoresis, 26 (2005) 2034–2046
MS spectrum of sialylated glycopeptides associated with the Fc region of the antibody. The lower inset represent the charge state of different glycopeptides, while the upper inset represent the tandem MS spectra of each of the glycopeptides. Structures of the glycan attached to the peptide backbones are represented by the following symbols: ( ) GlcNAc; ( ) fucose; ( ) mannose; ( ) galactose; ( ) N-acetylglycolylneuraminicacid.
Analysis of GlycopeptidesESI/MS
Ion-trap mass spectrum of human IgG tryptic digest averaged between 18-21 min. Inset, MS2 recording of m/z 1399 glycopeptide.
1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abu
ndan
ce
1399
.12
1317
.68
1480
.22
1500
.26
1141
.98
1264
.18
1186
.50
1507
.77
1581
.47
1449
.77
1641
.65
1245
.84
1376
.89
EEQYNSTYR EEQYNSTYR
EEQYNSTYR
1297
.18
EEQYNSTYR
EEQYNSTYR
EEQYNSTYR
EEQYNSTYR
EEQYNSTYR
EEQYNSTYR
EEQYNSTYR
EEQYNSTYR
EEQYNSTYR
EEQYNSTYR
1326
.47
1346
.6 1406
.57
1425
.57
1552
.03
EEQYNSTYR
HexNAc
HexNAc
FucHex
Hex
HexNAc
Hex
1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abu
ndan
ce
1399
.12
1317
.68
1480
.22
1500
.26
1141
.98
1264
.18
1186
.50
1507
.77
1581
.47
1449
.77
1641
.65
1245
.84
1376
.89
EEQYNSTYREEQYNSTYR EEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYREEQYNSTYR
1297
.18
EEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYREEQYNSTYR
EEQYNSTYREEQYNSTYREEQYNSTYR
1326
.47
1346
.6 1406
.57
1425
.57
1552
.03
EEQYNSTYREEQYNSTYREEQYNSTYR
HexNAc
HexNAc
FucHex
Hex
HexNAc
Hex
VETISFSFSEFEPGNDNLTLQGAALITQSGVLQLTK
VETISFSFSEFEPGNDNLTLQGAALITQSGVLQLTK
IRMPD
ECD/IRMPDMS3
K. Håkansson, M. J. Chalmers, J. P. Quinn, M. A. McFarland, C. L. Hendrickson,A. G. Marshall, Anal. Chem. 75, 3256-3262 (2003).
=Xylose=Fucose=Mannose=N-Acetyl Glucosamine
Analysis of GlycopeptidesFT MS (ECD & IRMPD)
Rel
ativ
e A
bund
ance
m / z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400
50
45
40
35
30
25
20
15
10
5
[M + 2H]2+
[GlcNAc - H2O]+
[Man2 - H2O]+
ν3
[GlcNAcMan - H2O]+
[M - Man6]2+
∗
[GlcNAcMan2 - H2O]+
[M - Man5]2+
[M - GlcNAcMan7]+
[M - Man4]2+
[Glc
NAc
Man
3-H
2O]+
[M - Man3]2+
[M - Man2]2+
[M -
Man
7]+
[M - Man]2+
NLTK
Trypsin digested
Adamson, J.T.; Håkansson, K. J. Proteome Res. 2006, 5, 493-501 .
Analysis of GlycopeptidesFT MS (IRMPD)
50
45
30
20
10
40
35
25
15
5
Rel
ativ
e A
bund
ance
m / z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
[M + 7H]7+
∗∗
∗ ∗
[GlcNAc - H2O]+
ν3
y’2+
b52+
[GlcNAcMan - H2O]+
b62+
b3+
[b8 - 2H2O]2+
b102+
b112+
b5+
[M - Man2]7+
[M - Man - H2O]7+
[M - Man]7+
R QHMDSSTSAA SSSNYCNQMM-b3 b5b6 b8 b10 b11
KSRNLTKDRC KPVNTFVHEy2
Adamson, J.T.; Håkansson, K. J. Proteome Res. 2006, 5, 493-501 .
Analysis of GlycopeptidesFT MS (IRMPD)
Glu C digested
50
45
30
20
10
40
35
25
15
5
Rel
ativ
e A
bund
ance
m / z200 300 400 500 600 700 800 900 1000
z2+• ν3 c’2+
∅z3
+•∅ c’3+ c’82+
z4+•c’92+
∗
c’102+
c’4+
c’112+
z5+•c’12
2+
c’132+
z112+
c’244+
∅
c’142+
z6+•
z122+•
y’122+
c’6+
c’152+
a223+•
z7+•
[M + 7H]7+
∅ ∅
∅
z152+•
c’243+
∅c’172+
c’8+
c’305+c’39
6+
∗
R QHMDSSTSAA SSSNYCNQMM KSRNLTKDRC KPVNTFVHE
Adamson, J.T.; Håkansson, K. J. Proteome Res. 2006, 5, 493-501 .
Analysis of GlycopeptidesFT MS (ECD)
Analysis of GlycopeptidesESI/MS (Lectin Enrichment)
M. Madera, Y. Mechref, M.V. Novotny, Anal. Chem. 77(13) (2005) 4081-4090.
High-performance affinity chromatography setup coupled on-line to electrospray mass spectrometry, including a system for loading of proteins/glycoproteins (A); and elution and on-line analysis of proteins/glycoproteins (B).
Nano column C8/C18
Lectin microcolumn
Auxiliary pump
C8/C18 trapping column
Systempump
Waste
A
Lectin microcolumn
Systempump
Nano column C8/C18
C8/C18 trapping column
Auxiliary pump
Waste
B
Nano column C8/C18
Lectin microcolumn
Auxiliary pump
C8/C18 trapping column
Systempump
Waste
A
Lectin microcolumn
Systempump
Nano column C8/C18
C8/C18 trapping column
Auxiliary pump
Waste
B
Analysis of GlycopeptidesESI/MS (Lectin Enrichment)
Extracted ion chromatograms of fetuin glycopeptides analyzed by on-line SNA-lectin trapping and LC/MS. Lectin unbound fractions (a, c, and e), and lectin bound fractions (b, d, and f).
85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00 125.00 130.00 135T0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
x150
x150
97.6
2
x96
x12
119.
58
123.
14
x216
121.
58
123.
18
LCPDCPLLAPLNDSR
LCPDCPLLAPLNDSR
VVHAVEVALATFNAESNGSYLQLVEISR
VVHAVEVALATFNAESNGSYLQLVEISR
RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR
RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR
a
b
c
d
e
f
85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00 125.00 130.00 135T0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
x150
x150
97.6
2
x96
x12
119.
58
123.
14
x216
121.
58
123.
18
LCPDCPLLAPLNDSR
LCPDCPLLAPLNDSR
VVHAVEVALATFNAESNGSYLQLVEISR
VVHAVEVALATFNAESNGSYLQLVEISR
RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR
RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR
a
b
c
d
e
f
M. Madera, Y. Mechref, M.V. Novotny, Anal. Chem. 77(13) (2005) 4081-4090.
Analysis of GlycopeptidesESI/MS (Hydrophilic Enrichment)
Y. Wada, M. Tajiri, S. Yoshida, Anal. Chem., 76 (2004) 6560-6565.
Analysis of GlycopeptidesESI/MS (Hydrophilic Enrichment)
Y. Wada, M. Tajiri, S. Yoshida, Anal. Chem., 76 (2004) 6560-6565.
Glycan ReleaseEnzymatic
Table 2. Glycoprotein oligosaccharide-releasing enzymes.
Highlighted enzymes are most commonly used
R.A. O'Neill J. Chromatogr. A 720 (1996) 201-215
Glycan ReleaseEnzymatic
Unlike N-glycans, no endoglycosidases are reliably available for the release of O-linked oligosaccharides, with the partial exception of endo-α-N-acetylgalactosaminidase, permitting the release of unsubstituted Core-1 O-glycans. However, this highly specific enzyme has very limited use, as it does not cover the other core structures. At this time, chemical release methods provide the only universal means for O-linked glycans.
Glycan ReleaseChemical
hydrazinolysisTakasaki and Kobata, Methods Enzymol., 50 (1978) 50.
improved hydrazinolysisPatel et al., Biochemistry, 32 (1993) 679.
Carlson β-elimination Carlson and Blackwell, J. Biol. Chem., 243 (1968) 616.
improved β-eliminationY. Huang, T. Konse, Y. Mechref, and M. V. Novotny, Rapid Commun. Mass Spectrom., 16 (2002) 1199-1204.
ammonia-based β-elimination Y. Huang, Y. Mechref and M.V. Novotny, Anal. Chem., 73 (2001) 6063.
Glycan ReleaseChemical (Hydrazinolysis)
N- and O-glycans can be chemically cleaved from glycoproteins with hydrazine (hydrazinolysis). O-glycans are claimed to be specifically released at 60oC, while 95oC is needed to release N-linked oligosaccharides. This chemical release approach suffers from several major disadvantages:
the reagent cleaves amidic bonds, including the linkage between the N-glycans and asparagine, the samples are destroyed. Consequently, any information is lost pertaining to the site of glycosylation and the extent to which it occurs. the acyl groups of N-acetylamino sugars and sialic acids are hydrolyzed, calling for a reacetylation step, assuming that a sialic acid possessed originally an acetyl group rather than any other substitution. the residual hydrazide or amino groups are often incorporated to the reducing terminus of some glycans. a loss of the reducing terminal GlcNAc is commonly observed as a result of conducting the reaction at high temperatures. It is essential to maintain strictly anhydrous conditions, which may not be always feasible.
Standard reagent: 1 M NaBH4 , 0.05 M NaOH
Reductive β-Elimination of O-Glycans
O OH
NH C CO
H
CH
ONAc
HO
OHHO
OHNH C CO
CH
H (or CH3)
H (or CH3)
NAcHO
OHHOOH
CH O
NAcHO
OHHO
OH
+
NaOH
NaBH4
CH2
Drawbacks:Oligosaccharides are reduced to alditols: lack of a reducing end
not feasible for chromatography Desalting is necessary: high concentration of salts
Glycan ReleaseChemical (β-Elimination)
Reductive β-Elimination with BH3.NH3 (ammonia borane complex)
Glycan ReleaseChemical (β-Elimination with BH3.NH3 )
O
NH C CO
H
CH
ONAc
HO
OHHO
OHNH C CO
CH
H (CH3)
H (CH3)
NAcHO
OHHO
OH
CH O
+
NAcHO
OHHOOH
CH2OH
BH3.NH3
NH3/H2O
Y. Huang, T. Konse, Y. Mechref, and M. V. Novotny, Rapid Commun. Mass Spectrom., 16 (2002) 1199-1204.
O
NH C CO
H
CH
ONAcHO
OHHO
OHNH C CO
CHH (or CH3)
H (or CH3)
NAcHO
OHHOOH
CH O
+NH3/H2O
Reducing-oligosaccharide
O
NAcHO
OHHO
OH
O
NAcHO
OHHO
NHCO3NH4
Glycosylamine-carbonate
O
NAcHO
OHHO
NH3 H2O
NH4HCO3 H2O
H3BO3
H3BO3
OH
glycoprotein Peptide
Glycosylamine
Reducing-oligosaccharide
O
NAcHO
OHHO
NH2
Glycan ReleaseChemical (ammonia-based β-Elimination)
Y. Huang, Y. Mechref and M.V. Novotny, Anal. Chem., 73 (2001) 6063.
Glycomics
Glycomics, or glycobiology is a discipline of biology that deals with the structure and function of oligosaccharides (chains of sugars). The term glycomics is derived from the chemical prefix for sweetness or a sugar, "glyco-", and was formed to follow the naming convention established by genomics (which deals with genes) and proteomics (which deals with proteins). The identity of the entirety of carbohydrates in a cell, a tissue, or an organism is thus collectively referred to as the glycome.
Since the ion yield and crystal formation in MALDI/MS analysis are adversely influenced by the presence of salts and buffers, their prior removal becomes desirable. Carbohydrates are generally less tolerant than proteins to salts and other compounds. This is despite the fact that small amounts of sodium or other alkali metals are required for efficient ionization. Many methods have been developed for the removal of salts and buffers.
drop dialysisNafion-117 membranes synthetic membranes (polyethylene and polypropylene)ion-exchange or hydrophobic resins packed pipette tipsHydrophobic resins
Glycomic AnalysisSample Preparation
Glycomic AnalysisSample Preparation (Packed Pipette Tip)
www.harvardapparatus.com
Glycomic AnalysisSample Preparation (Packed Pipette Tip)
www.harvardapparatus.com
Glycomic AnalysisSample Preparation (SPE)
www.harvardapparatus.com
Positive-ion MALDI mass spectra of the N-linked oligosaccharides derived from 1 mg of ribonuclease B:
(a) before PNGase F digestion; (b) enzymatic digest without purification; (c) enzymatic digest treated with C18 packing; and (d) enzymatic digested treated with the SP20SS resin.
Y. Huang, Y. Mechref, J. Tian, H. Gong, W. J. Lennarz and M. V. Novotny, Rapid Commun. Mass Spectrom. 14 (2000) 1233–1237.
Glycomic AnalysisSample Preparation
DHB (most commonly used matrix for MALDI-MS analysis of glycans) typically crystallizes as long needle-shaped crystals that originate at the periphery of the spot and project toward the center when a mixture of acetonitrile or methanol and water is used. An amorphous mixture of the analyte, contaminants and salts are present in the central region of the spot. It is known that in a mixture of glycans and glycoproteins, glycans were fractioned in the central region of the spot while the glycoproteins were in the periphery, as concluded from the acquired spectra. Therefore, a more even film of crystals is produced by re-dissolving the spot in dry ethanol and allowing it to recrystalize. In addition to producing a thin and even film of crystals, this technique also increased sensitivity by an order of magnitude as a result of more efficient mixing of matrix and analyte from a single solvent.Even film of crystals is also attained by drying under vacuum.
Glycomic AnalysisMS-based Approaches (MALDI-MS)
Glycomic AnalysisMS-based Approaches (MALDI-MS)
Matrices
Table 2. Matrices and additives utilized for the analysis of N-linked glycans.
matrix abbreviation structure
2,5-dihydroxybenzoic acid 2,5-DHB
2-hydroxy-5-methoxybenzoic acid ___
arabinosazone ara
1-hydroxyisoquinoline HIQ
3-aminoquinoline 3-AQ
5-chloro-2-mercaptobenzothiazole CBMT
COOHOH
HO
COOHOH
CH3O
N
OH
N
NH2
N
SSH
Cl
N
N
NNH
HC
HC
CH2OH
OH
OH
H
Glycomic AnalysisMS-based Approaches (MALDI-MS)
Matrices
Table 2. Continued.
matrix abbreviations structure
ferulic acid ___
2,5-dihydroxyacetophenone DHA
6-aza-2-thiothymine ATT
2,4,6-trihydroxyacetophenone THAP
OHOCH3
COOH
OH
OHHO
O
N
NN
OH
CH3
HS
O
OHHO
OH
While mass determination through MALDI/MS can often lead to compositional data (in terms of isobaric monosaccharides) additional information must be secured through other methodologies.Monosaccharide sequences, branching and, in some cases, linkages can be determined through fragmentation that a glycan may experience in either a post-source decay (PSD) or a collision-induced dissociation. The combination of MALDI/MS and enzymatic sequencing using exoglycosidases provides the necessary information related to sequence, branching and linkage of a glycan.
Glycomic AnalysisMS-based Approaches (MALDI-MS)
B1 C1 B2 C2 B3 C3
Z0Y0Z1Y1Z2Y2
A. Glycosidic FragmentationA. Glycosidic Fragmentation
OOH
OH
CH2OH
O
CH2OH
OH
OH
OHO
OOH
OH
CH2OH
O O R
Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.
Glycomic AnalysisFragmentation of Glycans in MALDI-MS
B. CrossB. Cross--ring Fragmentationring Fragmentation
0,2A1
1,5X13,5X2
2,4A22,5A3
OOH
OH
CH2OH
O
CH2OH
OH
OH
OHO
OOH
OH
CH2OH
O O R
Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.
Glycomic AnalysisFragmentation of Glycans in MALDI-MS
Glycomic AnalysisFragmentation of Glycans in MALDI-MS
Y. Mechref, A. G. Baker, M. V. Novotny, Carbohydrate Res., 313 (1998): 145–155.
Glycomic AnalysisFragmentation of Glycans in MALDI-MS
9.0 271.6 534.2 796.8 1059.4 1322.0Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
22.9
1036
.4
1257
.552
4
1123
.5
671.
2
849.
3
799.
3
475.
2
1095
.4
907.
3265
447.
2
73.3
226.
1
41.1
653.
2
164.
9 347.
1
527.
2
244.
1 1061
.4
771.
3
874.
3
509.
255
0.2
979.
3621
429.
2
583.
2
272.
1
185.
1
625.
2
399.
2
689.
2
147.
8
327.
230
6.9
1156
.467
3
745.
3
953.
3788
203.
1
817.
3
365.
1
712.
2
1010
.396
8
91.4
1182
.481
6
933.
3752
607.
2
1,5 X
4x,3β
0,2 A
53,
5 X4x
,3β
B4
C4
1,5
1,5 XX
33 αα
Y2
C3
C2α
B2α
Ion DB3/Y3β
B3
Y3α
Y4x
2,4 A
5
0,2 A
4
3,5 A
4
3,5
3,5 AA
33
0,4
0,4 AA
33
Z2
1,5 X
2
(Hex)2
1,5 X
1
Y1
Z1
B 4/Y
4x,3β
3,5X4α’
0,40,4AA33
3,53,5AA33
3,5 A
5
3,5A53,5X4α”
3,5X3β
1,51,5XX33αα
1,4A5
0,2A5
1,5X4α’
1,5X4α”
1,5X3β
0,2A4
3,5A4
1,51,5XX22
2,4A5
Na+
B1
C1
1,5 X
0
655.
2B 3
/Z 3
β
OCH2OH
OH OOH
OH
O
CH2OH
OH O
O
CH2OH
OH
NH
OH
CCH3
O
O
O
CH2OH
OH
NH
CCH3
O
O
O
O
CH2
OCH2OH
OH
OH
OH
OH
OH
OH
OO
CH2OH
OH OH
OH
Yehia Mechref, Milos V. Novotny and Cheni Kirshnan, Anal. Chem., Anal. Chem., 75 (2003), 4895-4903.
ProteinProtein
Sialic acid
Galactose
N-Acetylglucosamine
Mannose
PNGase F
N-Acetyl-β-D-glucosaminidase
β-Galactosidase
Neuraminidase
Glycomic AnalysisEnzymatic Sequencing (MALDI-MS)
ProteinProtein
Cou
nts
m/z
[M+Na]+
Glycomic AnalysisEnzymatic Sequencing (MALDI-MS)
Cou
nts
m/z
[M+Na]+-582
Glycomic AnalysisEnzymatic Sequencing (MALDI-MS)
Cou
nts
m/z
-582-324
Glycomic AnalysisEnzymatic Sequencing (MALDI-MS)
-324
Cou
nts
m/z
-406
Glycomic AnalysisEnzymatic Sequencing (MALDI-MS)
-582
N-glycosidase FNeuraminidase
β -Galactosidase
N-Acetyl-β-D-glucosaminidase
MALDI PLATE
Glycomic AnalysisEnzymatic Sequencing (MALDI-MS)
Y. Mechref, M.V. Novotny, Anal. Chem., 70 (1998) 455-463.
934.
477
1138
.52
1299
.92
1342
.02
1503
.08
1665
.55
1706
.78
1340
.52
1502
.55
1544
.17
1664
.58
1705
.79
1749
.58
1841
.92
1868
.05
1908
.31
1663
.85
2029
.07
2395
.03
-40000
-30000
-20000
-10000
0
10000
20000
1000 1500 2000 2500 3000 Mass (m/z)
Y. Mechref, L. Zidek, W. Ma, and M.V. Novotny, Glycobiology, 10 (2000) 231-235.
andand
andand
andand
Sequencing of N-Glycans Derived from Glycosylated MUP of the House Mouse
Glycomic AnalysisEnzymatic Sequencing (MALDI-MS)
Relative abundance [%]
Glycan [M+Na]+
(monoisotopic)NMRa CGEb LC/MSc MALDI-MS
(DHB)MALDI-MS
(ARA)
Man5 1257.423 57 51.5 51.9±2.0 50.9 ±1.2 57.8 ±1.1
Man6 1419.476 31 30.3 32.1±2.4 27.4 ±0.7 27.2 ±0.7
Man7 1581.529 4 4.0 6.3±0.6 7.3 ±1.1 5.4 ±0.4
Man8 1743.582 7 8.5 7.2±0.8 10.9 ±0.6 7.5 ±0.7
Man9 1905.634 1 3.7 1.6±0.1 3.5 ±0.6 2.0 ±0.4
a NMR data from Fu et al. 1994b CGE data from Guttman et al. 1995c LC/MS data from Gennaro et al.1996
Glycomic AnalysisQuantification (MALDI-MS)
Permethylation of Oligosaccharides for MS Analysis --allows simultaneous analysis of neutral and sialylated structurespermits reversed-phase LC separation of permethylated structuresenhances MSMSSimplifies MSMS interpretation
Glycomic AnalysisPermethylation
Methylation of carbohydrates in dimethyl sulfoxide by mixing with powdered sodium hydroxide and methyl iodide
(Ciucanu I, Kerek F Carbohydr. Res. 1984, 131, 209-217)
Oxidative degradation and peeling reactions due to the high pH resulting from dissolving sodium hydroxide powder prior to liquid-liquid extractions
Glycomic AnalysisPermethylation
Capillary Column (500 μm i.d. x 10 cm) packed with sodium hydroxide powder
Glycomic AnalysisPermethylation
P. Kang, Y. Mechref, I. Klouckova M. V. Novotny, Rapid Commun. Mass Spectrom, 19 (2005), 3421-3428.
Glycomic AnalysisPlatform
Human Blood Serum
GLYCOMIC MAPS
Protein Extraction
Glycopeptides
Glycoproteins
N-GLYCANS
MALDI TOF / MS
N-Glycans
Reduction & Alkylationwith TFE
Tryptic Digestion
PNGase F Digestion
Solid Phase Extraction
Permethylation
Human Blood Serum
GLYCOMIC MAPS
Protein Extraction
Glycopeptides
Glycoproteins
N-GLYCANS
MALDI TOF / MS
N-Glycans
Reduction & Alkylationwith TFE
Tryptic Digestion
PNGase F Digestion
Solid Phase Extraction
Permethylation
Glycomic AnalysisPlatform
849 1181 1513 1845 2177 3003 3507 4011 4515 5019Mass (m/z)
2792
.844
0
3242
.2
2605
.7
2967
.028
80.9
3603
.534
16.4
2676
.8
3777
.7
3055
.0
3691
.6
3331
.3
4052
.9
3212
.2
25090
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
2433
.3
1579
.9
2070
.1
1783
.9
2396
.0
1836
.0 2244
.1
1866
.01661
.9
1907
.0
1416
.8
2040
.1
1620
.9
1987
.0 2192
.221
12.2
2401
.0
2850
.9
4226
.1
4587
.5
N-Glycan Profile of Human Blood Serum of Breast Cancer Stage II Patient
Glycomic AnalysisFunctional Glycomics
isotope labeling with deuteromethyl iodide allows comparative glycomic profiles of two samples analyzed simultaneously.determines changes in abundances of glycans
Therefore, isotopic labeling will provide more precise comparison
Glycomic AnalysisComparative Glycomic Mapping (C-GlycoMAP)
Sample 1multi-step
sample1 preparation
multi-stepsample 2 preparation
permethylation
LIGHT
HEAVYCOMPARATIVE
GLYCOMIC MAPS
Sample 2
Glycomic AnalysisComparative Glycomic Mapping (C-GlycoMAP)
Glycomic AnalysisComparative Glycomic Mapping (C-GlycoMAP)
0
20
40
60
80
100
% In
tens
ity
4X
1500 1720 1940 2160 2380 2600Mass (m/z)
0
20
40
60
80
100 12X
0
20
40
60
80
100 1649
.3
1579
.9 1862
.5
1784
.0
2192
.2
1988
.1
2502
.0
2396
.3
12X 2288
.8
2075
. 7
1:6
1:1
6:1
Glycomic AnalysisComparative Glycomic Mapping (C-GlycoMAP)
C-GlycoMAP: In Breast Cancer Research
1499.0 1702 1906
3699 4308 4917
1499.0 2222.6 2946.2 3669.8 4393.4 5117.0Mass (m/z)
3000 3325 3650
Glycomic AnalysisChromatography-based Approaches
LC/MALDI/TOF-TOF MS of on-line permethylated glycans derived from a mixture of
glycoproteins.
Reversed-phase LC analysis of permethylated glycans 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
2222
21
20
19
1817
16151413
11
1098
7
65
12
34
Glycomic AnalysisChromatography-based Approaches
LC/ESI MS of on-line permethylated glycans
derived BSSL.
Reversed-phase LC analysis of permethylated glycans
Rel
ativ
e A
bund
ance
0 5 10 15 20 25 30 35 40 45 50 55 60
Time (min)
0
10
20
30
40
50
60
70
80
90
100
65 70
0.5 μg BSSL
Rel
ativ
e A
bund
ance
0 5 10 15 20 25 30 35 40 45 50 55 60
Time (min)
0
10
20
30
40
50
60
70
80
90
100
65 70
0.5 μg BSSL
Human BSSLNH2
SS SSS D H
Human BSSLNH2
SSSS SSSSS D H
COOH
Glycomic AnalysisChip-based Approaches (LC)
CHIP-LC/MS of Permethylated N-Glycans
0 10 20 30 40 50 600
1
2
3
4
0 10 20 30 40 50 60
Time [min]
0
1
2
3
4
Inte
nsity
(x10
7 )
Ribonuclease B
α1-Acid Glycoprotein
Glycomic AnalysisChromatography-based Approaches
Nano-LC-ESI-MS of oligosaccharides released from KLH by PNGase F treatment.
(A) Base peak chromatogram (mass range m/z 700-2800). (B-O) Mass spectra obtained for the time windows indicated by horizontal bars in (A). Sodium adducts are presented with m/z and deduced monosaccharide composition. H, hexose; N, N-acetylhexosamine; F, fucose; P, pentose; , sodium adduct. No m/z values are given for proton and potassium adducts.
Hydrophilic Interaction Chromatography (HILIC)
M. Wuhrer, C.A.M. Koeleman, D.A. M., C.H. Hokke, Anal. Chem. 76 (2004) 833.
Glycomic AnalysisChromatography-based Approaches
Comparison of negative ion (a) capillary LC/MS versus (b) nano-LC/MS analysis of neutral O-linked oligosaccharides (5.5 ng) using graphitised carbon chromatography (base peak chromatograms). Combined mass spectra of the region where oligosaccharides were eluted are shown as inserts.
Nano-LC with Graphitized Carbon Packings
G.N. Karlsson, N.L. Wilson, H.-J. Wirth, P. Dawes, H. Joshi, N.H. Packer, Rapid Commun. Mass Spectrom 18 (2004) 2282.
μA kVSheathLiquid
HV connectionES needle
365 μm OD200 μm ID
CEC Column160 μm OD100 μm ID
Pt Electrode
A. Que, Y. Mechref, Y. Huang, J. A. Taraszka, D.E. Clemmer and M. V. Novotny, B. Anal. Chem., 75 (2003), 1684-1690.
CEC-ESI/FT MS
Glycomic AnalysisElectrokinetically-driven Approaches (CEC)
CEC/ESI/FTMS of O-Glycans Derived from Mucin
a
b
10.5 min
12 min
A. Que, Y. Mechref, Y. Huang, J. A. Taraszka, D.E. Clemmer and M. V. Novotny, B. Anal. Chem., 75 (2003), 1684-1690.
Glycomic AnalysisElectrokinetically-driven Approaches (CEC)
a
b
A. Que, Y. Mechref, Y. Huang, J. A. Taraszka, D.E. Clemmer and M. V. Novotny, B. Anal. Chem., 75 (2003), 1684-1690.
Glycomic AnalysisElectrokinetically-driven Approaches (CEC)
Fuc Gal GlcNAc GalNAc-ol449611
GlcNAcGalNAc-ol
449NeuGc
449.
1747
611.
2276
757.
2853
[M+Na]+
756.
2650
449.
1752
[M+Na]+
a b
A. Que, Y. Mechref, Y. Huang, J. A. Taraszka, D.E. Clemmer and M. V. Novotny, B. Anal. Chem., 75 (2003), 1684-1690.
Glycomic AnalysisElectrokinetically-driven Approaches (CEC)
Composition of O-Glycans Derived from Mucin
A. Que, Y. Mechref, Y. Huang, J. A. Taraszka, D.E. Clemmer and M. V. Novotny, B. Anal. Chem., 75 (2003), 1684-1690.
Glycomic AnalysisElectrokinetically-driven Approaches (CEC)
Mass electrochromatogram of a complex fraction of the O-linked glycans chemically released from human bile salt-stimulated lipase. Conditions: amino column 28 cm, field strength 500Vcm–1, mobile phase acetonitrile/water/ammonium formate buffer (240 mM, pH 3.0, 55:44:1 v/v/v), injection 1 kV, 10 s 0
40000
80000
120000
0.0 4.2 8.2 12.2 16.2 20.0
Time (min)
TIC 1650
1796
1285
0
40000
80000
120000
0.0 4.2 8.2 12.2 16.2 20.0
Time (min)
TIC 1650
1796
1285
Glycomic AnalysisElectrokinetically-driven Approaches (CEC)
A. Que, M.V. Novotny, Anal. Bioanal. Chem. 375 (2003) 599.
singly charged
doubly charged
CEC/ESI/FTMS of OCEC/ESI/FTMS of O--Glycans Derived from BSSLGlycans Derived from BSSL
a
b
A. Que, Y. Mechref, Y. Huang, J. A. Taraszka, D.E. Clemmer and M. V. Novotny, Anal. Chem., 75 (2003), 1684-1690.
Glycomic AnalysisElectrokinetically-driven Approaches (CEC)
Composition of O-Glycans Derived from BSSL
A. Que, Y. Mechref, Y. Huang, J. A. Taraszka, D.E. Clemmer and M. V. Novotny, Anal. Chem., 75 (2003), 1684-1690.
Glycomic AnalysisElectrokinetically-driven Approaches (CEC)
T. J. Tegeler, Y. Mechref, K. Boraas, J. P. Reilly, M. V. Novotny, Anal. Chem., 76 (2004) 6698-6706.
CEC-MALDI MS
Glycomic AnalysisElectrokinetically-driven Approaches (CEC)
Glycomic AnalysisElectrokinetically-driven Approaches (CE)
Reductive amination of the oligosaccharide with APTS (8-aminopyrene-1,3,6-trisulfonate).
Labeling of Glycans by Reductive Amination
Glycomic AnalysisElectrokinetically-driven Approaches (CE)
CE profile of APTS-labeled glycans derived from mAb. The upper trace represents standard core-fucosylatedbiantennary/disialylated, monosialylated, and asialylatedglycans. Conditions: column, polyacrylamide-coated 50/365 mm ID/OD; length, 50.5 cm total, 40.5 cm effective length; temperature, 257C; injection pressure, 0.5 psi for 5.0 s; voltage, 15 kV anodic electroosmotic flow; lex 488 nm, lem 520.
10 12 14 16 18 20
0
1
2
3
4
5
RFU
t [min]
A2F
mAb
1
2 3
4
5 6 7
Y. Mechref, J. Muzikar, M.V. Novotny, Electrophoresis, 26 (2005) 2034–2046
CE-LIF of APTS-labled glycans derived from human blood serum of stage IV breast cancer patients with short (A), intermediate (B) and long (C) survival time
A
C
B
Minutes
18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0
RFU
0
20
40
60
80
100
120
140
160
180
200
220
A
C
B
Minutes
18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0
RFU
0
20
40
60
80
100
120
140
160
180
200
220
Minutes
13 14 15 16 17 18 19 20 21 22 23
RFU
0
10
20
30
40
50
60
Baseline
24 weeks Treatment
Minutes
13 14 15 16 17 18 19 20 21 22 23
RFU
0
10
20
30
40
50
60
Baseline
24 weeks Treatment
CE-LIF of APTS-labled glycans derived from human blood serum of prostate cacnerpatients before (upper trace) and after (lower trace) treatment .
Glycomic AnalysisElectrokinetically-driven Approaches (CE)
Conditions: 22 cm effective length, 31 cm total length; 20 mM Hepes, 1 mMPhosphate buffer (pH 6.5); Field Strength, 750 V/cm; Peak Identities; 1) Man 5, 2) Man 6, 3-5) Man 7, 6-7) Man 8, 8) Man 9
Glycomic AnalysisElectrokinetically-driven Approaches (CE)
0
1
2
3
4
5
6
7
2 2.2 2.4 2.6 2.8 3 3.2 3.4
Time (minutes)
Rel
ativ
eFl
uore
scen
ce
1
2
34 5
6
7
8
0
1
2
3
4
5
6
7
2 2.2 2.4 2.6 2.8 3 3.2 3.4
Time (minutes)
Rel
ativ
eFl
uore
scen
ce
1
2
34 5
6
7
8
Glycomic AnalysisElectrokinetically-driven Approaches (Chip-CE)
Electropherogram of glycans from AGP separated on a microfluidic device. The spiral separation channel was 22 cm long and the electric field strength was 800 V/cm.
Schematic of microfluidic device with twelve separation units. The design has twelve sample (S) reservoirs and separation channels, and buffer (B) and waste (W) reservoirs are shared between adjacent units. The effluent from the separation channels is detected just above where the channels merge into a single analysis (A) reservoir.
B
B
BB
B
B
BW
W
WW
W
W
S
S
S S
SS
SS
S
S
S
S
Adetect
Glycomic AnalysisChip-based Approaches (CE)
(A) ME profiling of serum samples from a noncirrhoticchronic hepatitis patient (upper trace) and cirrhotic patient (lower trace). (B) Profiling of the same samples using the ABI377 gel-based DNA-sequencer. Symbols: , N-acetylglucosamine; ○, mannose; □, galactose; , fucose.
1
3
6
7 8 9
1 2
3
4 5 6
7 8 9
Chronic hepatitis
Chronic hepatitis
Cirrhosis
Cirrhosis
N. Callewaert, H. van Vlierberghe, A. van Hecke, W. Laroy, J. Delanghe, R. Contreas, Nature Med. 10 (2004) 429.
CONCLUSIONS
Structural analysis of glycoproteins has come a long way during the last decade in terms of sensitivity and information content. It is still a multimethodologicaltask.The current emphasis is on working with complex mixtures (biological fluids and tissue extracts) on-line –building complete analytical platforms for functional glycomics and glycoproteomics.In various searches for biomarkers, the glycomic approach could be an easier route to diagnostic and prognostic information, but high-sensitivity glycoproteomics is needed for explanations in terms of biochemistry and physiology.
Further Readings
Yehia Mechref, Milos V. Novotny “Miniaturized separation techniques in glycomic investigations” J. Chromatogr. B, 841 (2006) 65–78.Milos V. Novotny, Yehia Mechref “New hyphenated methodologies in high-sensitivity glycoprotein analysis” J. Sep. Sci., 28 (2005) 1956–1968.Yehia Mechref, Milos V. Novotny “Structural Investigations of Glycoconjugates at High Sensitivity” Chem. Rev. 102 (2002) 321-369.