Study of N-linked and O-linked...

Study of N-linked and O-linked glycans

Techniques in Glycobiology Hui Zhang

September 9, 2013

Analysis of glycans –  Glycan structure diversity – Methods of glycan analysis

•  Preparation of glycans for analysis •  Lectins •  Chromatography •  Mass spectrometry

March 26, 2014 2

Structure and Names of Common Monosaccharide

Varki, A. e. a., Essentials of Glycobiology. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1999.

CFG and Essentials for Glycobiology

5 Chapter 1, Figure 6

Common classes of human glycoproteins

Essentials of Glycobiology Second Edition

Control of Glycoconjugate Biosynthesis

March 26, 2014 6

Control of Glycan Structures

•  Expression and acDviDes of enzyme •  NucleoDde sugar availability •  KineDcs of transports •  Glycoprotein expression •  Availability of glycosylaDon sites •  Glycans are mixtures of variants (glycoforms) on core structures

March 26, 2014 7

8

Major Classes of N-Glycans

Essentials of Glycobiology

Second Edition

9

CH2OH �

O � HO�N � | �H�

C �O �

CH2�

O

NH �S/T

N �

C=O �

CH3�

N-acetylglucosamine to Asn (GlcNAcβ1-Asn)

H�H�

H�

H�

Ser/Thr!

GalNAc!

Ser/Thr!

Man!

Ser/Thr!

Fuc!

Ser/Thr!

GlcNAc!

Ser/Thr!

Glc!

Hyl! Hyp!Ser!

Gal! Xyl!

!!

A few more O-glycans……!

O-Xyl….precursor for GAGs…!

O-GlcNAc….cytosolic glycosylation!

O-GlcNAc on Notch extracellular domain (recent report)!

O-glycans

O-Glycosidic Linkage

α!

Ser!

GalNAc!

After Esko, J

O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)!!β-elimination!

O

OH

H

H

HO

H

O

NAcHH

OH

NH2CHC

H2C

O

GalNAc!!!α!

Study of glycans –  Glycan structure diversity –  Methods of glycan analysis


March 26, 2014 12

Glycan Release

•  Trypsin digesDon of protein •  EnzymaDc release of N-‐glycans •  β-‐eliminaDon for O-‐linked glycans

– resulDng in release of glycan structure from hydroxyl of Ser or Thr

– ReducDon with NaBH4 prevents re-‐aSaching of glycan

Lance Wells

Treating Glycoproteins with Enzymes

Glycosidases: N-‐glycans Proteases (N-‐ and O-‐): Glycans can be obtained nonselecDvely by degradaDon of the protein by proteases to generate glycopepDdes.

March 26, 2014 15

Sigma

Enzymatic Release N-Glycans

R3=H or α1-3 fucose

Chemical Methods to Release Glycans

•  β-‐eliminaDon for O-‐linked glycans •  Hydrazinolysis: A chemical method that uses hydrazine to cleave amide bonds (e.g., the glycosylamine linkage between a sugar residue and asparagine or the acetamide bond in N-‐acetylhexosamines) to release both N-‐glycans and O-‐glycans or, under controlled condiDons, cleaves only the N-‐glycans.

•  Anhydrous hydrogen fluoride treatment: cleaves all the linkages of glycans while leaving pepDde bonds and glycopepDde linkage linkages of amino sugars intact

March 26, 2014 17

Analysis of glycans –  Glycan structure diversity –  Methods of glycan analysis


March 26, 2014 18

Lectins •  Carbohydrate-‐binding proteins

•  More than 2,000 lecDns

•  Many lecDns became commercially available

•  MulDple lecDns with disDnct binding specificiDes are used in combinaDon or in series

•  LecDn affinity –  ConA: mannosylaDon –  AAL: fucosylaDon –  SNA: α2-‐6 sialic acid

•  Sharon N, Lis H. History of lecDns: from hemaggluDnins to biological recogniDon molecules. Glycobiology. 2004 Nov;14(11):53R-‐62R.

•  hSp://proline.physics.iisc.ernet.in/cgi-‐bin/cancerdb/input.cgi •  hSp://nscdb.bic.physics.iisc.ernet.in

Chapter 45, Figure 1

Examples of types of N-glycan recognized by concanavalin A (Con A) from Canavalia ensiformis and Galanthus nivalis agglutinin (GNA)



Examples of types of N-glycan recognized by L-PHA, E-PHA, and DSA


March 26, 2014 22

Profile Global Glycosylation Changes Using Lectin Microarrays

p  94 lectins were immobilized on the array surface p  Lectin-antibody immunoassay p  Fluorescent detection

Profiling of Glycosylation of Target Glycoprotein Using High-density Lectin Array

Glass slide with N-Hydroxysuccinimide (NHS) esters coating

Lectin 1 Lectin 2 PSA PSA Human PSA protein extracted

from clinical specimens

Mouse anti-human PSA antibody

Rabbit anti-mouse IgG Alexa Fluor 647 conjugate

Yan Li, Sheng-Ce Tao, G. Steve Bova, Lori J. Sokoll, Daniel W. Chan, Heng Zhu, and Hui Zhang. 2010, Submitted

Glycomics of N-‐linked glycans –  Glycan structure diversity – Methods of glycan analysis


March 26, 2014 24

Separation of Glycans by Chromatography

•  Reverse phase column: separate pepDdes from glycans

•  Reverse phase LC: reducDve aminaDon is applied to increase the hydrophobicity

•  GraphiDzed carbon chromatography: separate structural isomers

•  Hydrophilic interacDon chromatography (HILIC) •  Glycan Analysis by High Performance Anion Exchange Chromatography

March 26, 2014 25

Separation of Glycans by Chromatography

•  Physico-‐chemical extracDon – Hydrophilic interacDon (HILIC) – Hydrophobic (C18 or graphiDzed carbon)

– Strong caDon exchange (SCX), Dtanium dioxide for sialylated glycans

March 26, 2014 26

Analysis of glycans –  Glycan structure diversity – Methods of glycan analysis


March 26, 2014 27

Characterization of Glycoproteins and Glycans Using MS

Glycoproteinsx

PepDdes +GlycopepDdes

Formerly glycosylated pepDdes + Glycans

Formerly glycosylated pepDdes

Glycans

Proteolysis

Release glycans

IonizaDon

Detector

Ion trap

Sample plate

Analyzer

FragmentaDon

SeparaDon

Sample preparation procedure MALDI-MS-MSn

Glycomics Using Mass Spectrometry

•  PutaDve structures are assigned to each molecular ion based on the usually unique glycan composiDon for a given mass.

•  Prior knowledge of biosynthesis.

March 26, 2014 29

Structure and Names of Common Monosaccharide

Varki, A. e. a., Essentials of Glycobiology. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1999.

162

291

146 162

203 162

203

132

Sep 9-13, 2012, International HUPO, Boston

•  Glycan extracDon

•  PotenDal issues –  Non-‐specific binding –  Sample loss (affinity; mulDple purificaDon) –  Difficulty to removal of reagents afer derivaDzaDon (sialic acid

modificaDon: reagents severely interfere glycan ionizaDon)

Current methods

Enzyme

C18/C8 Carbo modify Carbo MS

S. Yang and H. Zhang, Proteomics Clin. Appl. 2012, 11-12, 596-608

Sep 9-13, 2012, International HUPO, Boston

GIG (chemoselecDve method) Glycoprotein Immobilization for Glycan Extraction

(GIG)1

Immobilization on solid-phase: Immobilization in pH 10 on N-terminus and lysine

1S. Yang et al., Anal. Chem. 2013, 85(11), 5555-5561. 2P. Shah, S. Yang et al., Anal. Chem. 2013, 85 (7), 3606-3613.

3G.J. Rademaker et al., Anal. Biochem. 1998, 257, 149-160.

immobilize modify2

enzyme

β-elimination3

MS

wash

MS

Glycomics Using Tandem Mass Spectrometry

•  Assignments can be confirmed in a second experiment employing ESI-‐MS/MS instrumentaDon by selecDng each molecular ion for collisional acDvaDon and recording its fragment ion spectrum.

•  AddiDonal informaDon can be provided by MS experiments on chemical and enzymaDc digests, the choice of which is guided by the sequence informaDon provided by mass mapping and MS/MS experiments.

March 26, 2014 36

Glycan Fragmentation Ions

March 26, 2014 37

Joseph Zaia Mass Spectrometry and the Emerging Field of Glycomics. Chemistry & Biology (2008) 15, 881–892.

Domon and Costello, 1988

Linkage Analysis •  The principle of this method is to introduce a stable subsDtuent (an ether-‐linked methyl group) onto each free hydroxyl group of the naDve glycan.

•  The linkages, which are much more labile than the ether-‐linked methyl groups, are then cleaved with free hydroxyl groups at the posiDons that were previously involved in a linkage.

March 26, 2014 38

Glycan PermethylaDon •  Although MALDI-‐MS structural analysis of most glycans can basically be performed in their naDve forms.

•  DeterminaDon of branching, interglycosidic linkages and the presence of configuraDonal and conformaDonal isomers need permethylaDon.

•  PermethylaDon stabilizes glycans, yielding more predictable ion products when subjected to MS/MS experiments.

•  Permethylated glycans ionize more efficiently than their naDve counterparts.

March 26, 2014 39 Ciucanu I, Kerek F. Carbohydr. Res. 1984; 131: 209.

PermethylaDon of Glycans •  PermethylaDon of glycan-‐ OH→OMe

–  AddiDon of MeI •  Analyzed permethylated glycans by applying MSn fragmentaDon as

needed to completely determine the structure

March 26, 2014 40 J. Am. Chem. Soc. (2003) 125(52): 16213-9.


Glycosidases Used for Structural Analysis


Analysis of Sialylated glycans

•  Sialic acid are nine carbon containing acidic monosaccharides

•  Sialic acids are typically found at

the terminal residue of N-glycans, O-glycans, and glycosphingolipids

•  Sialic acids play crucial role in cell

surface interactions, protect cells from membrane proteolysis, help in cell adhesion, determine half life of glycoprotein in blood

Comparative Serum Glycoproteomics Using Lectin Selected Sialic Acid Glycoproteins with Mass Spectrometric Analysis: Application to Pancreatic Cancer Serum Journal of Proteome Research 2006, 5, 1792-1802

Mass Spectrometry of Sialic Acid

•  N-glycans are analyzed using MALDI

•  Sialic Acid are negatively charged

•  Sialic acid residues of sialoglycans are labile to the ionization process of MALDI TOF –  Insource Decay (labile carboxylic proton) –  Post Source Decay

(Matrix / LASER/ Detection Mode)

March 26, 2014 44

March 26, 2014 45

Mass Spectrometry of Sialic Acid

•  N-glycans are analyzed using MALDI

•  Sialic Acid are negatively charged

•  Sialicacid residues of sialoglycans are labile to the ionization process of MALDI TOF –  Insource Decay (labile carboxylic proton) –  Post Source Decay

(Matrix / LASER/ Detection Mode)

•  Several chemical derivatization methods have been employed for the MALDI analysis of sialylated glycans

•  Permethylation, Methyl esterification, Amidation

v

coupling

P-toludine

PNGaseF

Trypsin

MALDI MS LC MSMS

Shimadzu AXIMA Resonance Mass Spectrometric Analysis of Sialylated Glycans Using Solid Phase Labeling of Sialc Acids. PK Shah, S Yang, S Sun, P Aiyetan, KJ Yarema, and H Zhang. Analytical Chemistry (2013)

March 26, 2014 48

P-toluidine modified Sialic Acid Glycans on Solid-phase

Fetuin Glycans without Modificaiton

0

10

20

30

40

50

60

70

80

90

100

%Int.

2200 2400 2600 2800 3000 3200 3400m/z

13 mV[sum= 1309 mV] Profiles 1-100 Unsmoothed -Baseline 60

8.4.6a MS2 High Mass Gate Calibration Using ACTH(18-39)Data: punit\31jan\Fetuin Aniline power 95_2000_20001 1 Feb 2012 12:58 Cal: shadi_Nov3 3 Nov 2011 9:27 Shimadzu Biotech Axima Resonance 2.9.1.2.20100505: Mode positive, High 2000+, Power: 95

2653.5548{sn18}2340.6459{sn18}

2654.5462{sn16}

2966.4317{sn13}2339.6692{sn13}

2028.7383{sn12}

2341.6893{sn10} 2967.4612{sn9}2652.5462{sn9}

2288.6335{sn8}2026.7573{sn7}2968.3971{sn7}2342.6703{sn7}

2655.5447{sn6}2203.5944{sn6} 2516.4966{sn6}

2969.3603{sn5}2187.6296{sn4}

+4Na-3H

+3Na-2H +2Na-H

+Na

Matrix DHB +DMA

Reported N-‐glycans from Fetuin

Solid-phase permethylation of glycans for mass spectrometric analysis Rapid Commun Mass Spectrom. 2005 ; 19(23): 3421–3428

P-toluidine modified Fetuin Glycans

100

80

60

40

20

0 2000 2200 2400 2600 2800 3000 3200 3400 3600

Coupling

p-toluidine

PNGaseF/Trypsin

MALDI MS

Protein Amino Link Beads

P-Toluidine

P-Toluidine Heavy

Glycan LC-MS/MS

p-Toluidine-d9

Number of Sialic acids

Punit Shah et al. Analytical Chemistry. 2003

Serum Glycan Mixed 1:1 Light to heavy

Quantitative ?

Advantages of labeling •  It gives stability to the sialylated glycan

•  It removes negaDve charge from glycans

•  The label P-‐toludine is hydrophobic and allows retenDon on C18 columns •  The difference between the pair helps

–  DeterminaDon of number of sialic acid –  IdenDficaDon of glycans –  QuanDtaDon of sialylated glycans

•  Sample is first bound to the beads and hence the proteins afer removal of N glycans can be analyzed using TrypDc digesDon

•  Along with Sialic acid AsparDc acid and glutamic acid get modified and can be used for pepDde/protein QuanDtaDon

Site Mapping and Characterization

N

N

S

N

G Q M P

S S

S

S Q

T

T

V

A C L G

I C H

E

R

K

R

S K

G

E

A F D Y

P

P

M L

W

I

Lectin Affinity Capture, Isotope-coded Tagging and Mass Spectrometry to Identify N-linked Glycoproteins

Kaji H, et al. Nat Biotechnol 2003, 21(6):667-672.

Mass Spectra of Glyco and Non-glycopeptides After Releasing N-glycans

Identification and Quantification of N-linked Glycoproteins Using Solid-Phase Extraction of Glycopeptides (SPEG)

Zhang H, Li XJ, Martin DB, Aebersold R Nat Biotechnol 2003, 21(6):660-666.

Proteolysis"

Oxidation"

Coupling"

Wash"

Release"

Isotope labeling"Hydrazide beads"Glycans"Oxidized glycans"Succinic anhydride"

��

Site Specific Glycan Analysis

N

N

S

N

G Q M P

S S

S

S Q

T

T

V

A C L G

I C H

E

R

K

R

S K

G

E

A F D Y

P

P

M L

W

I

(75%) (25%)

Glycoproteomics on Glycopeptides with Glycan Attached

•  Mass spectrometric analysis of glycopepDdes is made challenging by the differing chemical properDes of glycans and pepDdes.

•  The ulDmate goal of glycoproteomics is to quanDfy the site occupancy of glycosylaDon in the proteome and the structures of glycoforms at each site.

60

Jonas Nilsson et al. Enrichment of glycopeptides for glycan structure and attachment site identification. Nature Methods 6, 809 - 811 (2009)

MS Analysis of Glycopeptides CID: PreferenDal fragmentaDon of the glycan

moiety of glycopepDdes. ETD: Abundant pepDde backbone dissociaDon is

observed for glycopepDdes using ETD.

March 26, 2014 62

Published in: Eden P. Go; Hua-Xin Liao; S. Munir Alam; David Hua; Barton F. Haynes; Heather Desaire; J. Proteome Res. 2013, 12, 1223-1234. DOI: 10.1021/pr300870t Copyright © 2013 American Chemical Society

BSA-OGlcNAcpepmix-2nmol-CID-ETD-HCD #5157 RT: 44.73 AV: 1 NL: 4.53E6T: FTMS + c NSI d Full ms2 [email protected] [100.00-1325.00]

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

MH, 1313.48

y11, 1216.61

y10, 1129.55

MH-‐He

xNAc

,1110.58

y9, 1030.51

y11-‐He

xNAc

, 1013.54

y10-‐He

xNAc

, 926.50

y9-‐HHe

xNAc

, 827.43

b7, 827.43

y8-‐HexNAc

, 730.38

MH+

2 , 657.83

y7-‐HexNAc

, 631.31

MH-‐He

xNAc

+2, 555.80

y6, 544.28

y7, 834.40

S+HexNAc Δ290.12m/z

y5, 487.26 y10-‐He

xNAc

+2, 463.75

y9-‐HexNAc

+2, 414.22

y3, 329.19

b3, 284.16

b3-‐H

2O, 266.15

HexN

Ac+1, 204.09

HexN

Ac-‐H

2O+1, 186.07

b2-‐H

2O, 167.08

HexN

Ac-‐66+

1 , 138.05

He

xNAc

-‐78+

1 , 126.05

PSVPV S GSAPGR

y6

y7

GlcNAc

HexN

Ac-‐60+

1 , 144.06

HCD spectra

204.084 HexNAc+1

[C8H14O5N]+ 186.075

[C8H12O4N]+ 168.064

[C8H10O3N]+

144.064 [C6H10O3N]+

138.054 [C7H8O2N]+

126.054 [C6H8O2N]+

HCD (zoomed in) D

PGGSTPVSSANMM

GlcNAc

b2,

154.

548

201.

858

179.

024

172.

741

122.

112

239.

007

110 130 150 170 190 210 230 250 m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

BSA-OGlcNAcpepmix-2nmol-CID-ETD-HCD #5085 RT: 44.09 AV: 1 NL: 2.23E6T: ITMS + c NSI d sa Full ms2 [email protected] [50.00-1325.00]

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

MH, 1314.58

MH-‐H 2O,1297.53

z11, 1200.57

c11, 1155.37

z10-‐H 2O, 1094.79

y9, 1030.77

z8, 918.54

z7, 819.61

b6, 769.57

MH+

2 , 657.51

z7-‐HexNAc

, 616.57

z6, 529.60

z5, 472.66

z4, 385.51

y8+3, 312.50

y2, 232.33

S+HexNAc Δ290.01m/z

c11-‐He

xNAc

, 952.43

y11, 1216.62

z10, 1113.58

PSVPV S GSAPGR

z6

z7

GlcNAc

ETD spectra

The Informatics Challenges of Diverse Glycomic Data

•  Efforts to correlate large data sets obtained from glycomic,

transcriptomic, genomic, and proteomic studies have met with several challenges.

•  RepresentaDon of glycan chemical structures is difficult because of their complexity and branching paSerns. The use of single alphabet codes, as employed to describe nucleic acid and amino acid sequences, is not applicable to glycans.

•  The field is in need of a comprehensive bioinformaDcs plakorm that stores, integrates, and processes data from glycomic and other “omic” studies and disseminates them in a meaningful fashion via the Internet to the scienDfic community.

March 26, 2014 67

Databases and Bioinformatics Platforms

•  GlycoSuiteDB, Sweet, KEGG GLYCAN •  The ConsorDum for FuncDonal Glycomics (CFG) •  EuroCarbDB •  NaDonal Center for Glycomics and glycoproteomics •  Glycomod: all possible composiDons of a glycan structure •  GlycoPep DB: N-‐glycopepDde composiDonal assignment •  Cartoonist: automated annotaDon of N-‐glycan MALDI TOF mass

spectra with cartoons represenDng the most plausible glycan assemblies synthesized by mammals using 300 manually determined archetypes.

March 26, 2014 68

Databases and Bioinformatics Platforms

•  Peptoonist: automated idenDficaDon of N-‐glycopep-des using a combina-on of MS and MS/MS data

•  Glyco-‐Peakfinder: rapid assignment of glycan composiDons, is intended to be enDrely a de novo plakorm for composiDonal analysis

•  SysBioWare: carbohydrate assignment •  NCRR GlycomicsPortal •  SimGlycan •  Accurate Glycan Analyzer •  GlycoWorkbench •  Byonics’s glycopepDde search Engine

March 26, 2014 69

Take Home Points 1.  Enzymatic approaches for N-linked site mapping 2.  Beta-elimination chemical approaches for O-linked site

mapping 3.  Glycopeptide analysis requires glycomics and proteomics to

confine the search space and for structural determination 4.  MSn approaches work for direct glycopeptide analysis 5.  ExD (ETD, ECD, etc.) look promising for glycopeptides 6.  HCD-triggered ETD approaches for mining deep and can aid

identification

Acknowledgements

Lance Wells, University of Geogia Shuang Yang, Johns Hopkins Punit Shah, Johns Hopkins

Mass Spectrometry Core of NIH/NHLBI Programs of Excellence

in Glycosciences (PEG, Jerry Hart, Jenny Van Eyk, Kevin

Yarema, etc.)

Thank You

[email protected]

March 26, 2014 72

Study of N-linked and O-linked...

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