Lecture 10. Glycoproteomics: Mass Spectrometric Methods for Analyzing Glycoproteins and Glycans Mass Spectrometry in an “Omics” World ME.330.804

Hui Zhang D Department t t off P Pathology, th l Cli Clinical i l Chemistry Ch i t Division Di i i Johns Hopkins University hzhang32@jhmi edu [email protected]

Topics • Glycoprotein classes • Glycoprotein enrichments • Release of p peptides p and glycosite analysis using MS • Glycomic Gl i analysis l i using i MS

Glycoproteins • Common protein modification: over 1/2 of the mammalian proteins • Diverse biological processes such as immune response, cellular regulation, and cell signaling g g • Alterations in glycosylation patterns are linked to diseases Dube DH, Bertozzi CR. Glycans in cancer and inflammation--potential for therapeutics and diagnostics Nat Rev Drug Discov. diagnostics. Discov 2005 Jun;4(6):477 Jun;4(6):477-88. 88 Zhang, H. & Cotter, R.J. Glycoproteomics: New Technology Developments and Applications Provide Renewed Interest in Glycoproteins. Clinical Proteomics (2008).

Types yp of Glycoproteins y p • N-glycosylation N glycosylation To the Asn side chain of proteins containing the sequon Asn-XSer/Thr (where X is any amino acid except Pro)

• O-GalNAc O GalNAc glycosylation Begins with the addition of a N-acetylgalactosamine to the OH of specific Ser or Thr side chains

• O-GlcNAc modification N-acetylglucosamine addition to the oxygen of specific Ser or Thr side chains

• Glycosylphosphatidylinositol (GPI) • Proteoglycans: Glycosaminoglycans (GAG)

Structure and Names of Common Monosaccharide

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

Major Classes of N-Glycans

Essentials of Glycobiology Second Edition

6

O-GalNAc Glycans with Different Core Structures Tn antigen

GalNAcαSer/Thr

Sialyl-Tn antigen

Siaα2-6GalNAcαSer/Thr

Peter-Katalinic J. Methods Enzymol. 2005;405:139-71.

Complex O-GalNAc Glycans with Different Core Structures

Essentials of Glycobiology Second Edition

Chapter 9, Figure 2

O-linked Glycosylation

Esko, J. 9

O GlcNAc Modifications O-GlcNAc O-GlcNAc transferase O OH

OH

O OH O

O

+

OH

OH

UDP

O

O

+

OH O

NH

NH

UDP

UDP-GlcNAc

O-GlcNAcase

– Modification at Ser/Thr residues; – A ubiquitous and dynamic form of protein modification; – Present in cytosolic proteins and nuclear proteins; – Some S modification difi i sites i overlap l with i h phosphorylation h h l i sites; i – Protein interactions, signal transduction, glucose sensing; Implicated in insulin resistance, stress response, and regulation Courtesy of off proteosome’s t ’ functions. f ti

Yingming Zhao

Torres CR, Hart GW: J Biol Chem 1984, 259(5):3308-3317. Wang, Z. & Hart, G.W. Clinical Proteomics 4 (2008).

General Structure of GPI Anchors

Essentials of Glycobiology Second Edition

Chapter 11, Figure 1

Proteoglycans Consist of a Protein Core and One or More Covalently Attached Glycosaminoglycan chains

Essentials of Glycobiology Second Edition

Glycosaminoglycans Consist of Repeating Disaccharide Units

Essentials of Glycobiology Second Edition

Chapter 16, Figure 3

Keratan Sulfates Contain a Sulfated Poly-N-acetyllactosamine Chain

Essentials of Glycobiology Second Edition

Chapter 16, Figure 4

Topics • Glycoprotein classes • Glycoprotein enrichments • Release of p peptides p and glycosite analysis using MS • Glycomic Gl i analysis l i using i MS

Glycoprotein Characterization • Glycoprotein Identification • Glycosylation type: glycan classes and their conjugation sites • Glycan structures • Glycans on each glycosylation site • Glycosylation occupancy • Quantitation 16 November 28, 2012

Characterization of Glycoproteins and Glycans Using MS Glycoproteins Proteolysis Peptides +Glycopeptides

Analyzer

Separation

Enrichment Glycopeptides

Detector

R l Release glycans l Formerly glycosylated peptides + Glycans

Ion trap

Fragmentation

Ionization

Formerly glycosylated peptides

Glycans

Sample preparation procedure

Sample plate

MALDI-MS-MSn

Isolation of Glycopeptides 1. D 1 Detection t ti with ith specific ifi llectins ti or antibodies tib di 2. Chemical reactions with constituent monosaccharides-A monosaccharides A general method labeling glycans on proteins involves periodate oxidation followed by Schiff base formation with amine- or hydrazide-based probes. probes 3. Metabolic labeling with chemically reactive monosaccharides 4. Label in vitro using a purified using a purified glycosyltransferase 5. LC-based enrichments November 28, 2012

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1 Lectins 1. • Carbohydrate-binding y gp proteins • More than 2,000 lectins • Many lectins became commercially available • Multiple lectins with distinct binding specificities are used in combination or in series • • •

Sharon N, Lis H. History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology. 2004 Nov;14(11):53R-62R. http://proline physics iisc ernet in/cgi-bin/cancerdb/input cgi http://proline.physics.iisc.ernet.in/cgi-bin/cancerdb/input.cgi http://nscdb.bic.physics.iisc.ernet.in

Lectin Affinity Chromatography Acronym

Metal Ions Specificity

Elution

Binding

Con A

Ca2+ Mn2+

a-Man>a-Glc 0.1-0.5 M a- N-linked MeMan

SNA

-

Siaα2-6Gal or GalNAc G lNA

0.1-0.5 L t Lactose

UEA

-

Α-L-Fuc

0.1-0.5 M L- Fuc Fuc

α2-6-linked Si Sia

Geng, M.; Zhang, X.; Bina, M.; Regnier, F., Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests digests. J Chromatogr B Biomed Sci Appl 2001, 752, 752 (2), (2) 293-306 293 306. Xiong, L.; Andrews, D.; Regnier, F., Comparative proteomics of glycoproteins based on lectin selection and isotope coding. J Proteome Res 2003, 2, (6), 618-25.

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

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

Other Affinity Reagents

• Antibodies against glycans: Monoclonal antibodies against Lewis X antigen. (Baeckström D et al. J Biol Chem. 1991; 266: 21537-21547) • Glycoprotein receptors: Mannose-6-phosphate (M6P) receptors for M6P-motifs containing glycoproteins l t i (Sl (Sleatt DE ett all P Proteomics t i 2005 2005; 5 5: 1520–1532)

2.Identification and Quantification of N-linked Glycoproteins Using Hydrazide Chemistry and Mass Spectrometry Proteolysis

Oxidation

Coupling

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

Wash

Isotope labeling Release

Hydrazide beads y Glycans Oxidized glycans Succinic anhydride

3. Metabolic Incorporation of the N3-GlcNAc into Proteins C2H5 O O

C2H5 O

Extracellular

C2H5

OC2H5

O

HN

O

I t Intracellular ll l

HO

OH

GlcNAc Kinase

N N N

OH

O

HN

OH O O N N N

Phospho-NAcetylglucosamine Mutase

Courtesy of Yingming Zhao

UDP-GlcNAc pyrophosphorylase

O-GlcNAc transferase

OH

OH

O OH O

O

OH

OH

UDP

O O

O

OH NH

+

N3

N3

UDP-N3-GlcNAc

NH

O-GlcNAcase

+

UDP

Chemoselective Conjugation Between N3 and a Phosphine

O

+

Ph2 P

S

Probe

Phosphine

Staudinger reaction

O

Probes:  Biotin  Flourescent dyes  Solid beads

N H

N3: small, inert, uncharged, non-polar, air-stable, and abiotic.Courtesy of Science (2000), 287, 2007-2010. Sprung, R.; Nandi, A.; Chen, Y.; Kim, S. C.; Barma, D.; Falck, J. R.; Zhao, Y., Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. J Proteome Res 2005, 4, (3), 950-7. Nandi A, Sprung R, Barma DK, Zhao Y, Kim SC, Falck JR, Zhao Y:Global identification of O-GlcNAc-modified proteins. Anal Chem 2006, 78(2):452-458.

Yingming Zhao

Id ntifi ti n of Identification f O O-N N3-GlcNAc-modified Gl NA m difi d Proteins P t in OH

O-N3-GlcNAc

O

Conjugation

Affinity purified by Affinity-purified streptavidin-beads

HO OH

O HN

O

Biotin

2% SDS 8 M Urea Digestion

HPLC/MS/MS for protein identification Courtesy of Yingming Zhao

4. Carbohydrate-tags Via Chemo-enzymatic Labeling • Utilizes a genetically engineered galactosyltransferase • Incorporate I t ketone k t analogs l off galactose l t t cellular to ll l O-glycosylated proteins • Incorporate p a biotin label through g coupling p g with aminoxy-biotin • •

Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Exploring the O-GlcNAc proteome: direct proteins from the brain. Proc Natl Acad Sci U S A 2004;; 101: identification of O-GlcNAc-modified p 13132-13137. Khidekel N, Ficarro SB, Clark PM, Bryan MC, Swaney DL, Rexach JE, Sun YE, Coon JJ, Peters EC, Hsieh-Wilson LC. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem Biol. 2007; 3: 339-348.

Chemoenzymatic y Strategy gy for Identifying y g O-GlcNAcglycosylated Proteins from Cellular Lysates

Application of the Strategy Toward Crystallin a

b

c

d

e

(a) MS analysis revealed the tagged O-GlcNAc peptide. (b) MS2 spectrum of the precursor ion revealed the signature loss of the ketone ketonebiotin moiety. (c) MS3 analysis revealed the loss of the GlcNAc moiety. moiety (d) MS4 analysis generated additional y and b ions that were used to sequence the peptide. (e) Summary of the y and b fragment ions.

5 LC 5. LC-based based Enrichment • •

• • •

Hydrophilic interaction LC (HILIC): the hydrophilic nature of glycopeptides (Wada Y, Tajiri M,Yoshida S. Anal Chem 2004; 76: 6560-6565) Size exclusion chromatography: masses of N-glycans are larger than 1200 Da; thus, most N-glycopeptides could be enriched by size-exclusion chromatography (Alvarez-Manilla G et al. J Proteome Res 2006;; 5: 701-708)) Boronic acid: Boronic acid forms boronic diesters through reaction of geminal diols (Sparbier K et al. J Chromatogr B 2006; 840: 29–36) Strong cation exchanger (SCX): Glycopeptides with a terminal sialic acid can be enriched by LC on an SCX column (Lewandrowski U et al. Mol Cell Proteomics 2007; 6: 1933–1941) Titanium dioxide: Sialic acid binds TiO2 (Larsen MR et al al. Mol Cell Proteomics 2007; 6: 1778–1787)

Topics • Glycoprotein class • Glycoprotein enrichments • Release of p peptides p and glycosite analysis using MS • Glycomic Gl i analysis l i using i MS

Release of N-Linked Glycans Using PNGase F CH2OH

K/R / O O HO

HO

C H2

N C H

• PNGase conversion of Asn to Asp N

• Mass shift confirms that the peptide was glycosylated and localizes site of N-linked

S/T HO

glycosylation K/R

p and gglycans y with 18O water • Labelingg ppeptides

Cleavage converts Asn to Asp

on the glycosylation site

CH2OH

K/R O O NH2 HO

HO

HO C

C H2

D S/T

HO K/R

Reducing glycan and 18O labeling

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Mass Spectra p of Glyco y and Nonglycopeptides After Releasing N-glycans

Enzymatic Release N-Glycans

Sigma

Enzymatic Release of O-Glycans

Sigma

Chemical Release of O-Glycans

Release of GPI • Successful cleavage by GPI-specific GPI specific phospholipases can be assessed by subsequently analyzing samples by MS, because removal of the GPI anchor causes a shift hift iin molecular l l mass. Thi This iis a common di diagnostic ti method for identifying the presence of a GPI anchor on a protein of interest • Another method is to treat the GPI-anchored protein with nitrous acid, which cleaves the unsubstituted glucosamine residue that links the glycan to the phosphatidylinositol (PI).

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Enzymatic y Release of GPI Anchors

Cleavage sites of phospholipases. Phospholipase C cut just before the phosphate attached to the R3 moiety. moiety Vikipedia

38

Release of Proteoglycans gy • Proteoglycans typically contain more glycan than protein. They may be separated by agarose gel electrophoresis and by ion-exchange chromatography, which separates on the basis of the charge conferred by sulfate groups. • Treatment of proteoglycans with GAG lyases will produce a shift in mass to remove most of the glycan portion. • Antibodies that recognize the remaining structures (“stubs”) may be used in western analysis. The lyases cleave a 4,5 unsaturated uronic acid at the no reducing end end. Anti-“stub” Anti stub antibodies recognize the sulfation of the penultimate Nacetylglucosamine or N-acetylgalactosamine residue. November 28, 2012

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Treating Glycoproteins with Proteases N-Glycans and O-glycans can be obtained bt i d nonselectively l ti l by b d degradation d ti of the protein by proteases to generate glycopeptides. l tid

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Chemical Methods to Release Glycans • Hydrazinolysis: A chemical method that uses hydrazine to cleave amide bonds (e (e.g., g the glycosylamine linkage between a sugar residue and asparagine p g or the acetamide bond in Nacetylhexosamines) to release both N-glycans and O-glycans or, under controlled conditions, cleaves only l the h N N-glycans. l • Anhydrous hydrogen fluoride treatment: cleaves all th lilinkages the k off glycans l while hil lleaving i peptide tid b bonds d and glycopeptide linkage linkages of amino sugars intact November 28, 2012

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Topics • Glycoprotein classes • Glycoprotein enrichments • Release of p peptides p and glycosite analysis using MS • Glycomic Gl i analysis l i using i MS

Control of Glycoconjugate Biosynthesis

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Control of Glycan y Structures • • • • • •

Expression and activities of enzyme Nucleotide sugar availability Kinetics of transports Glycoprotein expression Availability of glycosylation sites Glycans are mixtures of variants (glycoforms) on a core structure

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M Mass S Spectrometric i A Analysis l i of f Glycans Gl

• Sample preparation/purification • Separation • Ionization

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Sample Preparation Strategies for Glycans

Joseph p Zaia Mass Spectrometry p y and the Emerging g g Field of Glycomics. Chemistry & Biology (2008) 15, 881–892. November 28, 2012

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S p Separation ti n of f Glycans Gl n f for MS An Analysis l i • Reversed phase LC/MS: reductive amination is applied pp to increase the hydrophobicity • Graphitized carbon chromatographychromatography MS: separate structural isomers • Hydrophilic interaction chromatography (HILIC) • Lectin L ti affinity ffi it November 28, 2012

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Glycomics y Using g Mass Spectrometry p y • Putative structures are assigned to each molecular q g glycan y composition p ion based on the usuallyy unique for a given mass. • Prior knowledge of N- and O-glycan biosynthesis. • Assignments can be confirmed in a second experiment employing ESI-MS/MS instrumentation by selecting g each molecular ion for collisional activation and recording its fragment ion spectrum. • Additional information can be provided by MS experiments i t on chemical h i l and d enzymatic ti di digests, t th the choice of which is guided by the sequence information provided by mass mapping and MS/MS experiments. November 28, 2012

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Glycan Fragmentation Ions

Domon and Costello, 1988

Joseph Zaia Mass Spectrometry and the Emerging Field of Glycomics. Chemistry & Biology (2008) 15, 881–892. November 28, 2012

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Data from a Glycomics Study of N-glycans from Mouse Kidney

Essentials of Glycobiology Second Edition

Chapter 47, Figure 6

Linkage Analysis • The p principle p of this method is to introduce a stable substituent (an ethery g group) p) onto each free linked methyl hydroxyl group of the native glycan. • The glycosidic linkages, which are much more labile than the ether-linked methyl groups, are then cleaved with free hydroxyl groups at the positions that were previously involved in a linkage. November 28, 2012

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Differentiation of Glycan Isomers Using Tandem MS Analysis of Permethylated Glycans

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Glycosidases Used for Structural Analysis

Essentials of Glycobiology Second Edition

Chapter 47, Figure 2

Quantitative Glycomics • Label-free: Permethylation and MS • Stable isotope labels for glycomics: based on diff differential ti l stable t bl isotope i t labeling l b li (CH3I/CD3I) and d permethylation • Reductive Amination Labeling: d0/d4 pyridyl amine (PA)

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Determination of Anomericity y • Sequential q exoglycosidase gy digestions: g Cleavage by α- or β-exoglycosidases y of specific p indicates the anomericity terminal sugar residues. • Many glycosidases are specific for both monosaccharide residue and linkage type, allowing detailed structural conclusions, although the number of such enzymes available is limited. November 28, 2012

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Glycoproteomics: y p Analysis y of Glycopeptides with Glycan Attached • Mass spectrometric analysis of glycopeptides is made challenging by the differing chemical properties of glycans and peptides peptides. • The ultimate goal of glycoproteomics is to quantify p y of g glycosylation y y in the p proteome the site occupancy and the structures of glycoforms at each site.

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Enrichment of Glycopeptides y p p and MS Analysis • Lectins for affinity capture • Graphitized carbon solid phase extraction • Abundant peptide backbone dissociation is observed for glycopeptides using ETD. ETD CAD results in preferential fragmentation of the glycan moiety of glycopeptides glycopeptides. November 28, 2012

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

The Informatics Challenges of Diverse Glycomic Data • Efforts to correlate large data sets obtained from glycomic, l i transcriptomic, t i t i genomic, i and d proteomic t i studies t di have met with several challenges. • Representation of glycan chemical structures is difficult because of their complexity and branching patterns. The use of single alphabet codes, as employed to describe nucleic acid and amino acid sequences sequences, is not applicable to glycans. p bioinformatics • The field is in need of a comprehensive platform that stores, integrates, and processes data from glycomic and other “omic” studies and disseminates them in a meaningful fashion via the Internet to the scientific community. November 28, 2012

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Databases and Bioinformatics Platforms • • • • • • •

GlycoSuiteDB, y , Sweet,, KEGG GLYCAN The Consortium for Functional Glycomics (CFG) EuroCarbDB National Center for Glycomics and glycoproteomics Glycomod: all possible compositions of a glycan structure G GlycoPep DB: N-glycopeptide compositional assignment Cartoonist: automated annotation of N-glycan MALDI TOF mass spectra with cartoons representing the most plausible glycan assemblies synthesized by mammals using 300 manually determined archetypes. November 28, 2012

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Databases and Bioinformatics Platforms • Peptoonist: p automated identification of Nglycopeptides using a combination of MS and MS/MS data • Glyco-Peakfinder: Gl P kfi d rapid id assignment i t off glycan l compositions, is intended to be entirely a de novo platform for compositional analysis • SysBioWare: carbohydrate assignment • NCRR GlycomicsPortal • SimGlycan • Accurate Glycan Analyzer • GlycoWorkbench Gl W kb h November 28, 2012

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Summary • Glycoprotein classes • Glycoprotein enrichments • Release of peptides and glycosite analysis by MS • Glycomics y analysis y