Lecture 10. Glycoproteomics: Mass Spectrometric Methods ... · Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem Biol. 2007; 3: 339-348.

Lecture 10. Glycoproteomics: Mass Spectrometric Methods for Analyzing

Glycoproteins and Glycans

Mass Spectrometry in an “Omics” World ME.330.804

Hui ZhangD t t f P th l Cli i l Ch i t Di i iDepartment of Pathology, Clinical Chemistry Division

Johns Hopkins Universityhzhang32@jhmi [email protected]

TopicsTopics

• Glycoprotein classes• Glycoprotein enrichments• Glycoprotein enrichments• Release of peptides and p p

glycosite analysis using MSGl i l i i MS• Glycomic analysis using MS

Glycoproteins

• Common protein modification: over 1/2 of theCommon protein modification: over 1/2 of the mammalian proteins

• Diverse biological processes such as immune response, cellular regulation, and cell signalingg g

• Alterations in glycosylation patterns are linked to diseasesto diseases

Dube DH, Bertozzi CR. Glycans in cancer and inflammation--potential for therapeutics and diagnostics Nat Rev Drug Discov 2005 Jun;4(6):477 88diagnostics. Nat Rev Drug Discov. 2005 Jun;4(6):477-88.

Zhang, H. & Cotter, R.J. Glycoproteomics: New Technology Developments and Applications Provide Renewed Interest in Glycoproteins. Clinical Proteomics (2008).

Types of Glycoproteinsyp y p

• N-glycosylationN glycosylationTo the Asn side chain of proteins containing the sequon Asn-X-Ser/Thr (where X is any amino acid except Pro)

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

• O-GlcNAc modificationN-acetylglucosamine addition to the oxygen of specific Ser or Thrside chainsside chains

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

Structure and Names of Common MonosaccharideStructure 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-GlycansMajor Classes of N-Glycans

6Essentials of Glycobiology

Second Edition

O-GalNAc Glycans with Different Core Structures

Tn antigen GalNAcαSer/ThrTn antigen GalNAcαSer/ThrSialyl-Tn antigen Siaα2-6GalNAcαSer/Thr

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

Complex O-GalNAc Glycans with Different Core Structures

Chapter 9, Figure 2Essentials of Glycobiology

Second Edition

O-linked GlycosylationO-linked Glycosylation

9

Esko, J.

O GlcNAc ModificationsO-GlcNAc Modifications

O-GlcNAc transferaseO

UDP+ + UDPNH

O

O

OH

OH

OHO

NH

O

O

OH

OH

OHO

Modification at Ser/Thr residues;O-GlcNAcase

UDP-GlcNAc

+ UDPNHO

– Modification at Ser/Thr residues;– A ubiquitous and dynamic form of protein modification; – Present in cytosolic proteins and nuclear proteins;

S difi i i l i h h h l i i– Some modification sites overlap with phosphorylation sites;– Protein interactions, signal transduction, glucose sensing;Implicated in insulin resistance, stress response, and regulation f t ’ f tiof proteosome’s functions.

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

Courtesy of Yingming Zhao

General Structure of GPI Anchors


Second Edition

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


Second Edition

Keratan Sulfates Contain a Sulfated Poly-N-acetyllactosamine Chain


Second Edition

TopicsTopics



Glycoprotein CharacterizationGlycoprotein Characterization

• Glycoprotein Identification• Glycosylation type: glycan classes andGlycosylation type: glycan classes and

their conjugation sites• Glycan structures• Glycan structures• Glycans on each glycosylation site• Glycosylation occupancy• Quantitation

November 28, 201216

Characterization of Glycoproteins and Glycans Using MSGlycans Using MS

GlycoproteinsGlycoproteins

Peptides +Glycopeptides

Proteolysis

Analyzer Separation

R l lDetector

Analyzer Separation

Glycopeptides

Enrichment

Formerly glycosylated peptides + Glycans

Release glycans

Ionization

Ion trap Fragmentation

Formerly glycosylated peptides

Glycans Sample plate

Sample preparation procedure MALDI-MS-MSn

Isolation of GlycopeptidesIsolation of Glycopeptides

1 D t ti ith ifi l ti tib di1. Detection with specific lectins or antibodies2. Chemical reactions with constituent

monosaccharides-A general method labeling glycansmonosaccharides A general method labeling glycans on proteins involves periodate oxidation followed by Schiff base formation with amine- or hydrazide-based probesprobes.

3. Metabolic labeling with chemically reactive monosaccharides

4. Label in vitro using a purified using a purified glycosyltransferase

5. LC-based enrichmentsNovember 28, 2012 18

1 Lectins1. Lectins• Carbohydrate-binding proteinsy g p

• 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 ChromatographyLectin Affinity Chromatography

Acronym Metal Ions Specificity Elution BindingAcronym Metal Ions Specificity Elution Binding

Con A Ca2+ a-Man>a-Glc 0.1-0.5 M a- N-linkedMn2+ MeMan

SNA - Siaα2-6Gal G lNA

0.1-0.5 L t

α2-6-linked Sior GalNAc Lactose Sia

UEA - Α-L-Fuc 0.1-0.5 M L-Fuc

FucFuc

Geng, M.; Zhang, X.; Bina, M.; Regnier, F., Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests J Chromatogr B Biomed Sci Appl 2001, 752 (2) 293-306of glycopeptides from tryptic digests. J Chromatogr B Biomed Sci Appl 2001, 752, (2), 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 Capture, Isotope-coded Tagging and y p , p gg gMass Spectrometry to Identify N-linked Glycoproteins

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

Other Affinity ReagentsOther Affinity Reagents

• Antibodies against glycans: Monoclonal antibodies against Lewis X antigen. (Baeckström D et al. J BiolChem. 1991; 266: 21537-21547)

• Glycoprotein receptors: Mannose-6-phosphate (M6P) receptors for M6P-motifs containing l t i (Sl t DE t l P t i 2005 5glycoproteins (Sleat DE et al Proteomics 2005; 5:

1520–1532)

2.Identification and Quantification of N-linked GlycoproteinsUsing Hydrazide Chemistry and Mass SpectrometryUsing Hydrazide Chemistry and Mass Spectrometry

ProteolysisProteolysis

Oxidation

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

Coupling

Wash

Release

Isotope labeling

Hydrazide beadsGlycansRelease yOxidized glycansSuccinic anhydride

3. Metabolic Incorporation of the N3-GlcNAc into Proteins

I t ll lExtracellular

O

OC2H5

C H Intracellular

HN

O

O

OH

HO

OHO

OH

GlcNAc

HN

O

O

O

OOC2H5

C2H5

N N N

C2H5

HN O

N N N

GlcNAcKinase

Phospho-N-Acetylglucosamine

Mutase

N N N

O-GlcNAc transferase

Mutase

UDP-GlcNAcpyrophosphorylase


O-GlcNAc transferase

+ + UDP

OO H

O H

O HOUDP

N H

O

O

O H

O H

O HO

O-GlcNAcaseUDP-N3-GlcNAc

+ + UDPN HOO H

N 3

UDPN 3

Chemoselective Conjugation Between N3 and a Phosphine

Phosphine

Probe+ SPh2P

O

Probes:

Staudinger reaction

Biotin Flourescent dyes Solid beads

NH

O

N3: small, inert, uncharged, non-polar, air-stable, and abiotic. H

Courtesy of Yingming ZhaoScience (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.

Id ntifi ti n f O N Gl NA m difi d P t in

Conjugation Affinity-purified by

Identification of O-N3-GlcNAc-modified Proteins

O

OH

OHHO

O

O-N3-GlcNAcConjugation Affinity purified by

streptavidin-beads

Digestion

HN O

Biotin

2% SDS8 M Urea

Digestion

HPLC/MS/MS for protein identification


4. Carbohydrate-tags Via Chemo-enzymatic Labeling

• Utilizes a genetically engineered galactosyltransferaseI t k t l f l t t ll l• Incorporate ketone analogs of galactose to cellular O-glycosylated proteins

• Incorporate a biotin label through coupling with p g p gaminoxy-biotin

• Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci U S A 2004; 101: 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 Strategy for Identifying O-GlcNAc-y gy y gglycosylated Proteins from Cellular Lysates

a

Application of the Strategy Toward Crystallina

(a) MS analysis revealed the tagged O-GlcNAc peptide.

b (b) MS2 spectrum of the precursor ion revealed the signature loss of the ketone-

c

signature loss of the ketonebiotin moiety.

(c) MS3 analysis revealed the loss of the GlcNAc moiety

d

loss of the GlcNAc moiety.(d) MS4 analysis generated

additional y and b ions that dwere used to sequence the peptide.

(e) Summary of the y and be

(e) Summary of the y and b fragment ions.

5 LC based Enrichment5. LC-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• 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 MolTitanium dioxide: Sialic acid binds TiO2 (Larsen MR et al. Mol Cell Proteomics 2007; 6: 1778–1787)

TopicsTopics

• Glycoprotein class• Glycoprotein enrichments• Glycoprotein enrichments• Release of peptides and p p


Release of N-Linked Glycans Using PNGase FRelease of N-Linked Glycans Using PNGase F

CH OH /

• PNGase conversion of Asn to Asp• Mass shift confirms that the peptide was

CH2OH

NH

C

O

CH2

O

HON

K/R

Mass shift confirms that the peptide wasglycosylated and localizes site of N-linked glycosylation

• Labeling peptides and glycans with 18O water

HO

HO

2HO

S/T

K/R g p p g yon the glycosylation siteCleavage converts

Asn to AspCH2OH K/R

HO

NH2 C

O

CH2

O

HODHO

32

HO

S/T

K/R

Reducing glycan and 18O labeling

Mass Spectra of Glyco and Non-p yglycopeptides 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 phospholipasesSuccessful cleavage by GPI specific phospholipasescan be assessed by subsequently analyzing samples by MS, because removal of the GPI anchor causes a hift i l l Thi i di tishift in molecular mass. This is a common diagnostic

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 unsubstitutedglucosamine residue that links the glycan to theglucosamine residue that links the glycan to the phosphatidylinositol (PI).

November 28, 2012 37

Enzymatic Release of GPI Anchorsy

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

Vikipedia 38

before the phosphate attached to the R3 moiety.

Release of Proteoglycansg y

• 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.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 Anti-“stub”unsaturated uronic acid at the no reducing end. Anti stub antibodies recognize the sulfation of the penultimate N-acetylglucosamine or N-acetylgalactosamine residue.


Treating Glycoproteins with Treating Glycoproteins with Proteases

N-Glycans and O-glycans can be bt i d l ti l b d d tiobtained nonselectively by degradation

of the protein by proteases to generate l tidglycopeptides.


Chemical Methods to Release Glycans• Hydrazinolysis: A chemical method that uses

hydrazine to cleave amide bonds (e g thehydrazine to cleave amide bonds (e.g., the glycosylamine linkage between a sugar residue and asparagine or the acetamide bond in N-p gacetylhexosamines) to release both N-glycans and O-glycans or, under controlled conditions, cleaves

l h N lonly the N-glycans.• Anhydrous hydrogen fluoride treatment: cleaves all

th li k f l hil l i tid b dthe linkages of glycans while leaving peptide bonds and glycopeptide linkage linkages of amino sugars intactintact


TopicsTopics



Control of Glycoconjugate BiosynthesisControl of Glycoconjugate Biosynthesis


Control of Glycan Structuresy

• 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


M S i A l i f GlMass Spectrometric Analysis of Glycans

• Sample preparation/purification• Separation• IonizationIonization


Sample Preparation Strategies for Glycans

Joseph Zaia Mass Spectrometry and the Emerging Field of


p p y g gGlycomics. Chemistry & Biology (2008) 15, 881–892.

S p ti n f Gl n f MS An l iSeparation of Glycans for MS Analysis

• Reversed phase LC/MS: reductive amination is applied to increase the pphydrophobicity

• Graphitized carbon chromatography-Graphitized carbon chromatographyMS: separate structural isomers

• Hydrophilic interaction chromatography• Hydrophilic interaction chromatography (HILIC)L ti ffi it• Lectin affinity


Glycomics Using Mass Spectrometryy g p y• Putative structures are assigned to each molecular

ion based on the usually unique glycan composition y q g y pfor 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 each molecular ion for collisional activation gand recording its fragment ion spectrum.

• Additional information can be provided by MS i t h i l d ti di t thexperiments on chemical and enzymatic digests, the

choice of which is guided by the sequence information provided by mass mapping and MS/MS experiments.


Glycan Fragmentation Ions

Domon and Costello, 1988


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

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


Second Edition

Linkage AnalysisLinkage Analysis• The principle of this method is to p p

introduce a stable substituent (an ether-linked methyl group) onto each free y g p)hydroxyl group of the native glycan.

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


Differentiation of Glycan Isomers Using Tandem MS Analysis of Permethylated Glycans Tandem MS Analysis of Permethylated Glycans


Glycosidases Used for Structural Glycosidases Used for Structural Analysis


Second Edition

Quantitative GlycomicsQuantitative Glycomics

• Label-free: Permethylation and MS• Stable isotope labels for glycomics: based on

diff ti l t bl i t l b li (CH3I/CD3I) ddifferential stable isotope labeling (CH3I/CD3I) and permethylation

• Reductive Amination Labeling: d0/d4 pyridyl amineReductive Amination Labeling: d0/d4 pyridyl amine (PA)


Determination of Anomericityy

• Sequential exoglycosidase digestions: q g y gCleavage by α- or β-exoglycosidasesindicates the anomericity of specific y pterminal sugar residues.

• Many glycosidases are specific for bothMany glycosidases are specific for both monosaccharide residue and linkage type, allowing detailed structuraltype, allowing detailed structural conclusions, although the number of such enzymes available is limited.such enzymes available is limited.


Glycoproteomics: Analysis of y p yGlycopeptides with Glycan Attached

• Mass spectrometric analysis of glycopeptides is made challenging by the differing chemical properties of glycans and peptidesof glycans and peptides.

• The ultimate goal of glycoproteomics is to quantify the site occupancy of glycosylation in the proteome p y g y y pand the structures of glycoforms at each site.

56

Enrichment of Glycopeptides and y p pMS Analysis

• Lectins for affinity capture• Graphitized carbon solid phaseGraphitized carbon solid phase

extraction• Abundant peptide backbone• Abundant peptide backbone

dissociation is observed for glycopeptides using ETD CAD resultsglycopeptides using ETD. CAD results in preferential fragmentation of the glycan moiety of glycopeptidesglycan moiety of glycopeptides.


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 DataDiverse Glycomic Data

• Efforts to correlate large data sets obtained from l i t i t i i d t i t diglycomic, transcriptomic, genomic, and proteomic studies

have met with several challenges. • Representation of glycan chemical structures is difficultRepresentation 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 is not applicablenucleic acid and amino acid sequences, is not applicable to glycans.

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


Databases and Bioinformatics Platforms

• GlycoSuiteDB, Sweet, KEGG GLYCANy , ,• 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 mostmass spectra with cartoons representing the most plausible glycan assemblies synthesized by mammals using 300 manually determined archetypes.


Databases and Bioinformatics Platforms

• Peptoonist: automated identification of N-pglycopeptides using a combination of MS and MS/MS dataGl P kfi d id i t f l• Glyco-Peakfinder: rapid assignment of glycan compositions, is intended to be entirely a de novo platform for compositional analysis

• SysBioWare: carbohydrate assignment• NCRR GlycomicsPortal• SimGlycan• Accurate Glycan Analyzer

Gl W kb h• GlycoWorkbenchNovember 28, 2012 61

SummarySummary• Glycoprotein classesGlycoprotein classes• Glycoprotein enrichments• Release of peptides and

glycosite analysis by MSglycosite analysis by MS• Glycomics analysisy y

Lecture 10. Glycoproteomics: Mass Spectrometric Methods ... · Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem Biol. 2007; 3: 339-348.

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