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

http://www.ncgg.indiana.edu/

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


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




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)


A. Dell and H. Morris, Science 291, 2001


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


Tryptic Digestion

PNGase F Digestion


Permethylation

1500 2200 2900 3600 4300 5000Mass (m/z)

Healthy

Breast Cancer Stage IV


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.


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


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.



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


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.



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


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


Capillarypump

Waste

C4 trappingcolumn


Sample

MARS Column

On-line microcolumn lectinaffinity chromatographywith high-temp RPHPLC

Collection ofthe fractions

Tryptic Digestion

Nano LC-MS/MS Database search Result

Lectinmicrocolumn


Capillarypump

Waste

C4 trappingcolumn


Lectinmicrocolumn


Capillarypump

Waste

C4 trappingcolumn


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.


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



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



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


EEQYNSTYREEQYNSTYR





1326

.47

1346

.6 1406

.57

1425

.57

1552

.03


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


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


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


Auxiliary pump

Waste

B

Nano column C8/C18

Lectin microcolumn

Auxiliary pump


Systempump

Waste

A

Lectin microcolumn

Systempump

Nano column C8/C18


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


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





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.

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.

http://en.wikipedia.org/wiki/Biology

http://en.wikipedia.org/wiki/Oligosaccharide

http://en.wikipedia.org/wiki/Sugar

http://en.wikipedia.org/wiki/Genomics

http://en.wikipedia.org/wiki/Gene

http://en.wikipedia.org/wiki/Proteomics

http://en.wikipedia.org/wiki/Protein

http://en.wikipedia.org/wiki/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 (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)


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


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.


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



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




Y. Mechref, A. G. Baker, M. V. Novotny, Carbohydrate Res., 313 (1998): 145–155.


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]+


Cou

nts

m/z

[M+Na]+-582


Cou

nts

m/z

-582-324


-324

Cou

nts

m/z

-406


-582

N-glycosidase FNeuraminidase

β -Galactosidase

N-Acetyl-β-D-glucosaminidase

MALDI PLATE


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)


andand

andand

andand

Sequencing of N-Glycans Derived from Glycosylated MUP of the House Mouse


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


Capillary Column (500 μm i.d. x 10 cm) packed with sodium hydroxide powder


P. Kang, Y. Mechref, I. Klouckova M. V. Novotny, Rapid Commun. Mass Spectrom, 19 (2005), 3421-3428.


Human Blood Serum

GLYCOMIC MAPS

Protein Extraction

Glycopeptides

Glycoproteins

N-GLYCANS

MALDI TOF / MS

N-Glycans


Tryptic Digestion

PNGase F Digestion


Permethylation

Human Blood Serum

GLYCOMIC MAPS

Protein Extraction

Glycopeptides

Glycoproteins

N-GLYCANS

MALDI TOF / MS

N-Glycans


Tryptic Digestion

PNGase F Digestion


Permethylation


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



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


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


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


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.


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

b



Fuc Gal GlcNAc GalNAc-ol449611

GlcNAcGalNAc-ol

449NeuGc

449.

1747

611.

2276

757.

2853

[M+Na]+

756.

2650

449.

1752

[M+Na]+

a b



Composition of O-Glycans Derived from Mucin



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


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.


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.


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 (CE)

Reductive amination of the oligosaccharide with APTS (8-aminopyrene-1,3,6-trisulfonate).

Labeling of Glycans by Reductive Amination


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 .


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


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.

introduction to analytical glycobiology

Documents

different proteins

site specific

soluble proteins

nuclear proteins

cell surface oligosaccharides

different detection

cell surface landscape

plasma membrane