-techniques in glycobiology- nhlbi cardiopeg – gerald w.hart, august 19, 2013 funded by nhlbi...

-Techniques in Glycobiology-NHLBI CardioPEG ndash Gerald WHart August 19 2013

Funded by NHLBI P01HL107153

Introduction to

GlycanAnalysis

Techniques in Glycobiology

2

Topics Overview of Glycoproteomic and Glycomic Approaches

Composition Analysis of Glycans ndash Labs 821 amp 94

Radiochemical and Bioorthogonal Labelling of Glycans ndash Lab 830

Detection of Glycoproteins in Polyacrylamide Gels

Lectin Arrays to Analyze Glycoproteins ndash Lab 1011

Antibody Detection of Specific Glycans in Gels

Detection of Specific Glyosphingolipids on TLC- Lab 107 amp109

Methods for Isolation of Glycoproteins

Enzymatic and Chemical Release of Glycans from Glycoproteins ndash Lab 823

Labeling Released Glycans ndash Lab 830

Methods to Separate Released Glycans ndashLab 911

Glycosidases amp Structural Analyses ndash Lab 918

Methylation Analysis for Linkage Determination ndash Lab 826

MALDI-TOF Profiling of Glycans ndash Lab 1014

Nuclear Magnetic Resonance Spectroscopy ndash Labs 1016 amp 1018

GPI-Anchors amp Glycosaminoglycans ndash Lab 925

Glycosyltransferases As Probes- Lab 9273

J Mass Spectrom 2013 48 627ndash639

Types of Glycoproteomics ndash Require Different Methods

4

NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 | wwwnaturecomnaturechemicalbiology

Many Options for Glycoprotein Analyses

5


Level of Analysis amp Approach is Determined by Biological Question

6

Rough Estimates of Glycans by Colorimetric Assays (Lab 1)

Wed August 21 2013

Phenol-Sulfuric Acid Assay for hexoses amp pentoses

MBTH Assay for Hexosamines amp Acetylhexosamines

Carbazole Assay for Uronic Acids

BCA Assay for Reducing Sugars

Analysis of Free Sialic Acids From Glycoconjugates

by the Thiobarbituric acid assay

7

Useful as a starting place to estimate the quantity of glycans in a tissue or cell

Compositional Analysis by HPAEC-PAD

8

HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection

Compositional Analysis by GC-MS of Alditol Acetates

9

Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine) GC-MS

Alditols 1 peak per monosaccharide

Chapter 47 Figure 1

Radiolabeling strategies for the detection of glycans

Essentials of Glycobiology Second Edition

10

Metabolic Labeling of Glycoproteins with Radioactive Sugars

Martin D SniderCurrent Protocols in Cell Biology (2002) 781-7811

Nice discussion of issues

1 Competition with glucose ndash need to compromise

bull it is advisable to use these Precursors (open squares) in medium with reduced Glc (01 mgml) Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis

bull cells must not exhaust the supply of Glc or other nutrients in the medium

bull ldquoCatch 22rdquo ndash need to lower glucose but you donrsquot want to alter glycosylation

11

Scott T Laughlin amp Carolyn R BertozziVOL2 NO11 | 2007 | NATURE PROTOCOLS

12

Analysis of glycans using the bioorthogonal chemical reporter strategy

Chapter 48 Figure 3

Metabolic and covalent labeling of glycans for in vivo imaging of the glycome


13Drawback is low efficiency of incorporation

Laughlin amp Bertozzi 12ndash17 PNAS January 6 2009 vol 106 no 1

14

The bioorthogonal chemical reporter strategy for imaging glycans


15

Imaging of Glycans in Living Zebrafish

Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans ndash Temporal resolution during development

Proteomics 2006 6 5385ndash5408

16

Detection of Glycoproteins in SDS-PAGE


17


ANALYTICAL BIOCHEMISTRY 71 223-230 (1976)

18


periodic acid-Schiff (PAS) staining

Early work on RBC Surface Glycoproteins

Clin Proteom (2009) 552ndash68

19

Pro-Q Emerald 488 glycoproteinstain ndash Proprietary dye reacts with periodate reacted glycans

SYPRORuby total protein stain

Selective Staining of Glycoproteins on 2D-Gels


Lectin Blotting is a Powerful Tool to Detect Glycoproteins


DSL Lectin

LEL Lectin AAL Lectin

UEA I Lectin

OMICS A Journal of Integrative BiologyVolume 14 Number 4 2010

22

O-GlcNAc is Abundant on Nuclear amp Cytosolic Proteins 23

Detection of Glycoproteins by Glycan-Specific Antibodies

Eg Pan-Specific antibody to O-GlcNAc

24

Detection of Glycosphingolipids by Antibodies After TLC

Anal Chem 2009 81 9481ndash9492


25

Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes

H Geyer R Geyer Biochimica et Biophysica Acta 1764 (2006) 1853ndash1869

26

Typical Protocol Used to Analyze Glycoproteins

Current Opinion in Chemical Biology 2007 1152ndash58

27

Affinity methods for isolation of glycoproteins


28

Unnatural sugars used in the isolation of glycoproteins

L R Ruhaak amp G Zauner amp C Huhn amp C Bruggink amp A M Deelder amp M WuhrerAnal Bioanal Chem (2010) 3973457ndash3481

Options for Releasing Glycans From Proteins

29

30

Endoglycosidases for N-Glycans

31

PNGase F works best on Denatured Proteins or Glycopeptides But Does Not Release All N-Glycans Equally

(PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)

Sigma-Aldrich Chemical Deglycosylation Strategies

32

Several Endo-Glycosidases For Releasing N-GlycansAre Available Commerically

PNGase F useful in Site Mapping by MS

33

Sigma-Aldrich Glycoprotein Handbook

Unlike PNGase F O-Glycosidase is Highly Specfic


34

Enzymatic De-Glycosylation of O-Glycopeptides Requires Multiple Enzymes

Alkaline szlig-Elimination releases SerThr-linked Sugars

GlcNAc

O|

[3H]Gal[3H]Gal(1-4)GlcNAcitol

-Elimination

NaOHNaBH4

Often done with borohydride to prevent lsquopeelingrsquo35

ACS CHEMICAL BIOLOGY VOL4 NO9 bull 715ndash732 bull 2009bull

Differential glycan analysis using isotope labeling and mass spectrometry

36

Alkaline induced-b Elimination

CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H

Strategy for O-GlcNAcO-Phosphate site mapping

0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF

Replacement of O-GlcNAc with DTTUsing b-eliminationmichael addition

CNH

C

O

CH2H

DTT (or BAP)

Michael Addition

HSCH2CHOHCHOHCH2SH (DTT)

1 Provides tag forAffinity enrichment

2 Tag is stable in massspectrometer

Relative intensity

Relative intensity

Relative intensity

BEMAD Site MappingWells et al Molecular amp Cellular Proteomics (2002)

37

Mix

Performic acid treatment

lt--Trypsin--gt

O-GlcNAc-peptides O-phosphate-peptides

Control Cells Experimental Cells

Alkaline Phosphatase

BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT

O-GlcNAcase O-GlcNAcaseAlkaline Phosphatase

LC-MSMS

Heavy and Light DTT labeled O-GlcNAc-peptides

Heavy and Light DTT labeled O-phosphate-peptides

Thiol Enrichment Thiol Enrichment

Johns Hopkins38

Combination of Enzymatic Tagging with lsquoClick-Itrsquo Chemistry amp Solid-Phase BEMAD to Map Sites

BEMAD replaces the labile modification with a stable nucleoplilic group

J Am Chem Soc 125 (52) 16162 -16163 2003

39


40

Hydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linked

oligosaccharides ndashDestroys the Peptide

Anhydrous Hydrazine = Rocket Fuel


Strategies for the analysis of released glycoprotein-glycans

41

Bayer EA et al Meth Enzymol 184 415 (1990)42

Periodate Oxidation to Label Glycans


Labeling of glycans A 2-Aminobenzoic acid (2-AA) labeling via reductive amination

B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition

C labeling with phenylhydrazide and

D glycan permethylation

43


44


45

46

Methods to Separate Released Glycans

Tools of Glycome Profiling

47

HPLC of Glycans

Carbohydrate Research 94 (1981) C7-CV

NormalPhaseChromatography

48

Normal-Phase Chromatography of Underivatized GlycansA Type of HILIC


49

Analysis of N-linked oligosaccharides from human 1-acidGlycoprotein - 2-AB-labeled oligosaccharides

HPAEC

RP-HPLC

Glycoprotein Analysis Manual Sigma-Aldrich

50

Normal Phase Chromatography of 2-AB labeled Glycans

Oligosaccharide Profiling of O- ‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2- ‐AA)Elisabeth A Kast and Elizabeth A Higgins - ‐ GlycoSolu1048991ons Corpora1048991on Worcester MA

51

Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia- based B- elimination at both 40C and 60C


52

Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans

RNAase B

Ovalbumin

Fetuin


53

Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorptionionization (MALDI)ndashFourier transform ion cyclotron resonance (FTICR)ndashMS


54

A natural glycan microarray approach with reductively aminated glycans

55

Ion-Exhange Chrom ndash Separate Glycans by ChargeDEAE-Sephadex Separation of Glycans with 1 2 or 3 Sialic acids

Size After Desialylation

Gel Filtration Chromatography ndash Separate Oligosaccharides by Size

56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase

Dionex with Pulsed-Amperometeric

Detection ndash A Breakthrough in the mid-80s

J Chromatogr A 720 (1996)295-321

At pH 13Carbohydratesionize and Become Charged molecules

57

Derivatization of Glycans for

Electrophoresis

58

Capillary Electrophoresis of Glycans

as published in BTi October 2004 59

Fluorophore-Assisted Carbohydrate Electrophoresis

60

From Glyko


(FACE)

61

Hydrophilic Interaction Liquid Chromatography (HILIC)

62

(Formally called ldquoNormal Phaserdquo Chromatography)


63

HPLC-HILIC profile of N-glycans released from heavy chainhuman serum IgG

64

Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases

Chapter 47 Figure 2

Glycosidases used for structural analysis


65


66

Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans

Chapter 47 Figure 3Essentials of Glycobiology

Second Edition

A simple example of methylation analysis showing a structural motif that may be found in the polysaccharide glycogen

67

Methylation Analysis is Used to Determine Linkages

Enzymes or NMR are used to Determine Anomericity

Methyl Ether isAcid Stable

100 RXN

Reduction EliminatesAnomeric Mixtures

Chapter 47 Figure 6

Data from a glycomics study of N-glycans from mouse kidney


68

AssumptionsBased UponKnown Pathways

Typically Confirmed byMSMS and enzymes

Current Opinion in Structural Biology 2009 19498ndash506

Example of MALDI-TOF Profiling of Glycans

69

Chapter 47 Figure 4

1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans


70

NMR is the mostPowerful MethodFor Glycan StructureDetermination

Provided 2 things are True

1 Enough Material

2 Oligosaccharide Is pure

Chapter 47 Figure 5

NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F


71

Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1 mz 962 CE = 31 28 26 26 MS conditions were as described for Figure 1 The oligosaccharide structure and fragmentation pathway are shown at the top of the figure

Published in Douglas M Sheeley Vernon N Reinhold Anal Chem 1998 70 3053-3059DOI 101021ac9713058Copyright copy 1998 American Chemical Society

Complete Structural Analyses of Glycans Requires Several Approaches MS NMR Enzymes Fractionation

Ion Trap MSA Powerful tool For Structural Determination of Glycans

72

73

GPI-Anchored Proteins Are Extracted into the Detergent PhaseUsing Two-Phase Separation with TritonX-114

74

Basic Structure of GPI-Anchors

75

Strategy for Analysis of GPI-Anchors

76

Chemical and enzymatic reactions of GPI anchors


Second Edition

77

Characterization of Glycosaminoglycans

Bacterial EliminasesAre Powerful Tools

1 Sequential Degradation Followed by Gel Filtration

2 Other Separation Methods

78

FACE Analysis of GAG-Derived Disaccharides

79

HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB

80

Glycosyltransferases as Probes

81

82

83

Galactosyltransferase Labeling of Living Cells O-GlcNAc

84

Conclusions1 There are now a multitude of powerful methods to

analyze glycoconjugates

2 However most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry

3 Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site

4 Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field

-Techniques in Glycobiology-


Topics

Slide 4

Slide 5

Slide 6

Rough Estimates of Glycans by Colorimetric Assays (Lab 1) Wed



Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

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Slide 34


Slide 36

Slide 37

Slide 38

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Slide 40

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Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54

Ion-Exhange Chrom ndash Separate Glycans by Charge DEAE-Sephadex

Gel Filtration Chromatography ndash Separate Oligosaccharides by Si

Dionex with Pulsed-Amperometeric Detection ndash A Breakthrough in

Derivatization of Glycans for Electrophoresis



Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)


Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

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Slide 84


2




















4



5



6


Wed August 21 2013







7



8



9



Chapter 47 Figure 1



10








11


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

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Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

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Slide 73

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Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84




















4



5



6


Wed August 21 2013







7



8



9



Chapter 47 Figure 1



10








11


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84



5



6


Wed August 21 2013







7



8



9



Chapter 47 Figure 1



10








11


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84



6


Wed August 21 2013







7



8



9



Chapter 47 Figure 1



10








11


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


Wed August 21 2013







7



8



9



Chapter 47 Figure 1



10








11


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


8



9



Chapter 47 Figure 1



10








11


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


9



Chapter 47 Figure 1



10








11


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84

Chapter 47 Figure 1



10








11


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84








11


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


12


Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84

Chapter 48 Figure 3





14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


14



15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


15




16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


16



17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


17



18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


18





19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


19







DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84




DSL Lectin


UEA I Lectin


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


22




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84




24




25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


25



26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


26



27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


27



28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


28




29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84



29

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84

30


31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84

31




32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


32



33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84

33




34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


34



GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


GlcNAc

O|


-Elimination

NaOHNaBH4




36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84



36


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


CNH

C

O

CH2

(Serine-O-GlcNAc)

(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H


0

100

0

100

0

100

13143

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

12474

14217

mz1100 1650

mz1100 1650

mz1100 1650

MALDI-TOF


CNH

C

O

CH2H

DTT (or BAP)

Michael Addition




Relative intensity

Relative intensity

Relative intensity


37

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84

Mix


lt--Trypsin--gt




BEMADHeavy DTT

LC-MSMS

Add Controls

BEMADLight DTT


LC-MSMS




Johns Hopkins38



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84



J Am Chem Soc 125 (52) 16162 -16163 2003

39


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


40






41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84



41








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84








43


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


44


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


45

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84

46



47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


47

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84

HPLC of Glycans



48



49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


49


HPAEC

RP-HPLC


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


50



51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


51



52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


52


RNAase B

Ovalbumin

Fetuin


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


53



54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


54


55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84

55




56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


56

Beta -Galase

Beta-Hexosaminidase

Alpha-Mannosidase

Beta-Mannosidase

Beta-Hexosaminidase

Alpha-Fucosidase



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84



J Chromatogr A 720 (1996)295-321


57


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84


Electrophoresis

58




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34


Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

HPLC of Glycans

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54









Slide 63

Slide 64

Slide 65

Slide 66

Slide 67

Slide 68

Slide 69

Slide 70

Slide 71

Slide 72

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Slide 78

Slide 79

Slide 80

Slide 81

Slide 82

Slide 83

Slide 84




60

From Glyko


(FACE)

61


62



63


64


Chapter 47 Figure 2



65


66



Second Edition


67




100 RXN


Chapter 47 Figure 6



68





69

Chapter 47 Figure 4



70



1 Enough Material


Chapter 47 Figure 5



71





72

73


74


75


76



Second Edition

77





78


79


80


81

82

83


84








Topics

Slide 4

Slide 5

Slide 6




Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

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HPLC of Glycans

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(FACE)

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HPLC of Glycans

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100 RXN


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HPLC of Glycans

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Chapter 47 Figure 2



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100 RXN


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HPLC of Glycans

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Chapter 47 Figure 2



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100 RXN


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Chapter 47 Figure 4



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HPLC of Glycans

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Chapter 47 Figure 2



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100 RXN


Chapter 47 Figure 6



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1 Enough Material


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HPLC of Glycans

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100 RXN


Chapter 47 Figure 6



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Chapter 47 Figure 4



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HPLC of Glycans

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100 RXN


Chapter 47 Figure 6



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HPLC of Glycans

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Chapter 47 Figure 6



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HPLC of Glycans

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Chapter 47 Figure 4



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HPLC of Glycans

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Chapter 47 Figure 4



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HPLC of Glycans

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Chapter 47 Figure 5



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HPLC of Glycans

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HPLC of Glycans

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HPLC of Glycans

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HPLC of Glycans

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HPLC of Glycans

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HPLC of Glycans

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HPLC of Glycans

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HPLC of Glycans

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HPLC of Glycans

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-techniques in glycobiology- nhlbi cardiopeg – gerald w.hart, august 19, 2013 funded by nhlbi...

Documents

glycan analysis slide

monosaccharide slide

quantity of glycans

labeling released glycans

chemical release of

covalent labeling of

detection of glycoproteins

compositional analysis