Characterization of Glycan Structure

Essentials in Glycobiology

June 1, 2004

Brad Hayes

Roles of Glycoprotein-associated Carbohydrates

Quality Control/folding.

(deglucosylation/reglucosylation)

Solubility.

Circulating half-life.

(LH, FSH, vWF, ASGPR)

Cell-cell interactions.

(lymphocyte homing)

Pharmaceutical Concerns Regarding Carbohydrates

Pharmacokinetics: Influence on receptor binding.

Pharmacodynamics: Distribution. Clearance.

Define product as “well-characterized”.

Lot-to-lot variability.

Definition of intellectual property.

Branched. Synthesis is not “template driven”. Alternative linkage positions are possible. Alternative anomeric configurations are possible. Cell-type specific glycosylation. Influence of environmental conditions.

[Glucose] [NH3] pH

Site-specific glycosylation. Microheterogeneity.

Carbohydrate Analysis Offers Unique Challenges

Considerations in Designing an Analytical Scheme

Question

ResourcesAmount of

Material

Workflow

Sample Prep

Separation

Detection

Purity

Release of Glycans

Enzyme Digestion

Salts

HPLC

CE

PAD

Fluorescence

MS

Analysis of Glycans Still Bound to Proteins

Is the protein of interest glycosylated?

Fluorescent detection of sialic acids (<1pMole).

Monosaccharide composition analysis.

If so, are there N-glycans or O-glycans or both?

Mannose vs. Galactosamine (GalNAc)

What is the contribution to molecular weight?

Monosaccharide composition on a per mg basis.

Compare monosaccharide to amino acid composition.

Composition Analysis

Sialic Acid Determination

Diverse family of molecules.

Humans make antibodies against animal proteins withNeuGc.

Sialylation depends on culture conditions.

Incomplete sialylation associated with increased clearance.

When sample limited:

Is my protein glycosylated?

Is there enough material for monosaccharide analysis?

When sample not limited:

Batch-to-batch variability for recombinant proteins.

Extent of deglycosylation for crystallogrphic analysis.

Assess diversity of sialic acids present.

Sialic Acid Determination

Release with mild acid (2 m HOAc, 80oC, 3 hrs.).

“specific” for sialic acid release

doesn’t remove modifications from NeuAc

Anaylze by HPAEC-PAD

sensitivity of ~200 pMoles

Analyze by RP- HPLC after fluorescent derivatization.

OPD

DMB (~ 1 pMole)

Analyze by GC-MS as heptafluorobutyrate derivative.

Sensitive Sialic Acid Determination

Morimoto et.al. Anal. Chem. 2001

0 5 10 15 20 25 30 35 40 45

Neu5Gc

Neu5Gc9Ac

Reagent

Neu5Ac

Neu5,7Ac2

Neu5,9Ac2

Neu5,7(8),9Ac3

Time (minutes)

Flu

ores

cen

ce

BSMSialics

Composition Analysis: Neutral and Amino Sugars

Is my protein glycosylated?

Are there N- or O-glycans?

Relative contribution to mass?

What are the likely structures?

0 5 10 15 20 2520

30

40

50

Time (minutes)

Fuc GalNH2

GlcNH2Gal

Glc

Man/Xyl

nC

Standards

Hydrolysis, dry, HPAEC-PAD

Analysis of a Human Protein

Monosaccharide nMoles

Galactose

0

Glucosamine

Galactosamine

Fucose

0.366

0.03

0.317

0.672

0.115

Glucose

Mannose/Xylose

NeuAc 0.558

Major Species Minor Species

33 66

3

6

Data Predicted Structures

No N-Glycans.

On average, only one O-glycan per protein molecule.

No NeuGc, as expected.

Composition Analysis: Neutral and Amino Sugars

CE analysis of APTS derivatized monosaccharides

GC-MS of TMS derivatives:

or alditol acetates

or heptafluorobutyrate

From Beckman-Coulter Primer 8 on CE

Chen and Evangelista Anal. Chem. 1995

www.ccrc.uga.edu/~rcarlson/Carbstr.pdf

Release of Glycans for Further AnalysisRelease of N-glycans:

PNGase F. Broad spectrum for release of N-glycans

blocked by core 1,3 Fuc or bisecting Xyl

PNGase A. core 1,3 Fuc or bisecting Xyl okay

Prefers smaller, neutral glycans

Endo H. Only if ManII has not yet acted

HydrazineRelease of O-glycans:

Alkaline induced -elimination. Can’t fluorescently tag

O-glycanase. Only Gal1,3GalNAc

HydrazineRelease of glycolipid glycans:

Endoglycoceramidase

Glycan Profiling

Need to consider separation with detection

Charge

Size

Heterogeneity

Comparative differences between samples

Profiling Glycans by HPAEC-PADDisadvantages:

Retention time only.Introduces salt.

Sialic acid modifications.

Advantages: Fast.

Few pMole sensitivity.Reproducible.

Time (minutes)20 30 40

0

10

20

nC

28.85 29.35Neu5Ac3Gal4GlcNAc2Man6

Man4GlcNAc4GlcNAc(-ol)Neu5Ac6Gal4GlcNAc2Man3Neu5Ac3Gal4GlcNAc4

Neu5Ac6Gal4GlcNAc2Man6Man4GlcNAc4GlcNAc(-ol)

Neu5Ac6Gal4GlcNAc2Man3Neu5Ac3Gal4GlcNAc4

NeuAc(2,3)Gal elute later than NeuAc(2,6)Gal

Gal(1,3)GlcNAc elute later than Gal (1,4)GlcNAc

Fucosylated elute earlier than non-fucosylated

Neu5Gc elute later than Neu5Ac

+/- Exoglycosidases

Fluorescent Derivatization

Anumula, K. Anal. Biochem. 2000

Profiling/Sequencing Tagged Glycans by NP-HPLC

Anumula and Dhume Glycobiology 1998 Guile et.al Anal. Biochem. 1996

Profiling/Sequencing Glycans by CE-LIF

+Hexosaminidase

+Hexosaminidase

+ 1-2,3 mannosidase

Ma and Nashebeh Anal. Chem. 1990

Profiling Glycans by MALDI-TOF Mass Spectrometry

Native glycansCharge heterogeneityLoss of sialic acid

matrix acidityPost-source decay

Fragmentation mainly at the glycosidic bondsPreferential cleavages limit structural information

Permethylated glycans“Neutralize” carboxyl groupsReduced desialylationEnhanced cross-ring cleavages

•Pulsed laser

Flight tube

High voltage

Simplified Schematic of a MALDI-TOF

With MALDI ionization get almost

exclusively singly charged species

ABUNDANCE

MASS (m/z)

10,000

5,0002431.68

2793.05

3242.52

3603.90

3692.02

3487.79

4053.39

4660.124228.86

or

4414.83

5324.88

4589.03

5572.175151.69

4834.30

Normal

2000 2800 3600 4400 5200 6000

8,000

2431.87

2793.05

3243.293866.59

3603.48

3692.49

4414.834227.81

3937.64

4053.72

4,000

5151.255324.38

5571.77

Patient

MALDI-TOF Profiling of Glycans in Disease

Sequencing of Glycans with Mass Spectrometry

Already touched on use of exoglycosidases with

separation either by HPLC or CE

and detection either by PAD or fluorescence

Can also use mass spectrometry for sequencing.

Mechref et.al. Carbo Res. 1998

Branching and Linkage Analysis

How the components are put together

Mechref et.al. Carbo Res. 1998

Sequencing of Glycans with Mass Spectrometry

Linkage Analysis with Mass Spectrometry

Ionization

Method

Mass Analyzer/

SelectorMALDI

ESI

Quadrapole

Ion Trap

Collision

Cell

Detector

Quadrapole

Ion Trap

TOF

Linkage Analysis with Mass Spectrometry

Sheeley and Reinhold Anal. Chem. 1998

Linkage analysis by GC-MS of partially methylated alditol acetates.

Methylate exposed hydroxyl groups.

Hydrolyze glycosidic bonds.

Reduce with borohydride.

Acetylate newly created hydroxyl groups.

Analyze by GC-MS.

Ab

un

dan

ce

14 16 18 20 22 24

ST6Gal-I null

time (minutes)

2Man1

Fuc1-

2,4Man1

2,6Man13,6Man1

14 16 18 20 22 24

Ab

un

dan

ce

ST6Gal-I wt

time (minutes)

3Gal1

6Gal1Gal1-

Linkage Analysis of Total N-Glycans from ST6Gal-I Deificient Mice by GC-MS

Linkage Analysis by NMR Spectroscopy

Sia3H3eq Sia6

H3eq

Sia3H3ax Sia6

H3ax

(+)

(–)

500 MHz nano-NMR spectra from total N-glycans from ST6Gal-I deficient mice .

MALDI-TOF Analysis of HPAEC Fractions

0 10 20 30 40 50 600

100

200

300

400

nC

Time (minutes)

1

2

45

910

1214

1617

20

31

43

44

45

46

2853.10Peak 44

7000

0

MASS (m/z)2000 2600 3200 3800 4400 5000

2853.10

MASS (m/z)2000 2600 3200 3800 4400 5000

6000Peak 45

ABUNDANCE

N-Glycans from ST6Gal-I deficient mouse liver

Heparan Sulfate/Heparin

(3,6SO3)GlcNSO3

(2SO3)IduA

(6SO3)GlcNSO3GlcNSO3GlcUA GlcNAc GlcUAGlcUA GlcNSO3

(2SO3)IduA

Chondroit in Sulfate/Dermatan Sulfate

GalNAc(2SO3)IduA

(4SO3)GalNAcGalNAcGlcUA GalNAc GlcUAGlcUA

(4SO3)(4SO3)(6SO3)GalNAcGlcUA(4SO3)

Structure of Sulfated Glycosaminoglycans

Analysis of Glycosaminoglycans

Isolate GAG/proteoglycan by ion exchange

chromatography.

Depolymerize enzymatically to generate disaccharides.

Fraction disaccharides (IP-RP HPLC, IEC HPLC).

Detect by UV (>100 pMoles).

Detect fluorescently following post-column derivatization (<10 pMoles).

Post-column Labeleing with 2-Cyanoacetamide

2-Cyanoactamide reacts with reducing sugars under basic conditions at high temperature.

Used as post-column derivatization for GAG disaccharide analysis.

Applicable to any separation.

Column

1%2-CA

0.25 MNaOH

Reaction

Coil

130oC

“Cooling

Bath”

UV Detector

Fluorescence

Detector

>100 pMole

>5 pMole

Flow Path for Post-column Labeling

IP-RP HPLC

IEC HPLC

0 10 20 30 40 50 60 70

Di-0S

Di-4SDi-6S

Di-UA2S

Di-diSE

Di-diSB

Di-diSD

Di-TriS

300

100

800

Di-0S

Di-4S

600

400

200

200

Di-0SDi-4S

Di-6S Di-diSE100

0

Control

Patient

Stds

0

Time (minutes)

Flu

ores

cen

ceIon-Pair Rerverse Phase HPLC

Analysis of Serum Chondroitin Sulfate

Real Name Short FromΔUA-GalNAc ΔDi-0S

ΔUA-GalNAc-4S ΔDi-4S

ΔUA-GalNAc-6S ΔDi-6S

ΔUA-GalNAc-4S-6S ΔDi-diSE

ΔUA-2S-GalNAc ΔDi-UA2S

ΔUA-2S-GalNAc-4S ΔDi-diSb

ΔUA-2S-GalNAc-6S ΔDi-diSd

ΔUA-2S-GalNAc ΔDi-triS

Chondroitinase Disaccharides

Results from ~10 l of serum

50 pMole of Heparin Disaccharides Stds

0

200

UA-[1,4]-GlcN

UA-[1,4]-GlcNAc

UA-[1,4]-GlcN-6S

UA-2S-[1,4]-GlcN

UA-[1,4]-GlcNS UA-[1,4]-GlcNAc-6S

UA-2S-[1,4]-GlcN-6S

UA-[1,4]-GlcNS-6S

UA-2S-[1,4]-GlcNS

UA-2S-[1,4]-GlcNS-6S

100

0 10 20 30 40 50 60 70

0

1000

UA-[1,4]-GlcNAc

UA-[1,4]-GlcNS

UA-[1,4]-GlcNAc-6S

UA-[1,4]-GlcNS-6SUA-2S-[1,4]-GlcNS

UA-2S-[1,4]-GlcNAc-6S

UA-2S-[1,4]-GlcNS-6S

Time (minutes)

Lyase Digest of 1 ug Authentic Heparin

Flu

ores

cen

ceIon Exchange Fractionation of Heparin-derived Disaccharides

UA-2S-[1,4]-GlcNAc-6S

Test for O-GlcNAcO-GlcNAc is a substrate for galactosyltransferase and can

be radiolabeled using UDP-[3H]galactose as the donor.

Protein-O-GlcNAc Protein-O-GlcNAc-[3H]Gal

UDP-[3H]Gal UDP

1,4 Galactosyl-transferase

O-GlcNAc is susceptible to alkaline-induced -elimination.

Protein-O-GlcNAc-[3H]GalNaOH

NaBH4

[3H]Gal1,4GlcNAcitol

.

Pit 1 Saccharides

Gal1,4GlcNAcitol

0

2

0

6

4

0

2

4

6

10 20 30

A

Elution Time

800

00 10 3020

400

B

1 32G

al1

,3G

lcN

Aci

tol

Gal1

,3G

alN

Aci

tol

Gal1

,4G

lcN

Aci

tol

Pit 1 is O-GlcNAcylated

Separation of disaccharide alditols on Dionex MA-1 column

Summary

The choice of approaches and technologies depends on the question asked and the depth of understanding required.

A complete structural characterization generally requires the use of several different and complementray techniques.

There are many options available for characterizing glycans.