SAMPLE PREPARATION AND CAPILLARY GEL

ELECTROPHORESIS PROFILING OF

ASPARAGINE LINKED GLYCANS

András Guttman

Horváth Laboratory of Bioseparation Sciences,

University of Debrecen, Hungary

Leopold-Franzens University, Innsbruck, Austria

PhyNexus Users Group Symposium, South San Francisco, CA, AUG 27, 2014.

Significance of Glycosylation

Glycans protect proteins, orient binding faces,

prevent non-specific interactions, increase protein

stability, e.g., N-glycans shield large areas of protein

surfaces from proteases.

Main glycosylation types on glycoproteins:

- N-linked (Asn)

- O-linked (Ser/Thr)

Microheterogeneity + site occupancy

biological activity changes

Glycocalyx at the surface of an erythrocyte (Taylor and Drickamer, Glycobiology, Oxford)

Asn Asn

Asn Asn Asn Asn

Ser/ Thr Ser/ Thr Ser/ Thr Ser/ Thr Ser/ Thr

Rillahan, C.; Paulson, J. Annu. Rev. Biochem. 2011, 80, 797-823.

Rillahan, C.; Paulson, J. Annu. Rev. Biochem. 2011, 80, 797-823.

For N-linked oligosaccharides, a 14-sugar precursor is

first added to the asparagine in the polypeptide chain

of the target protein. The structure of this precursor

is common to most eukaryotes, and contains 3

glucose, 9 mannose, and 2 N-acetylglucosamine

molecules. A complex set of reactions attaches this

branched chain to a carrier molecule (dolichol), and

then it is transferred to the appropriate point on the

polypeptide chain as it is translocated into the ER

lumen for further processing. After attachment, once

the protein is correctly folded, the three glucose

residues are removed from the chain and the protein

is available for export from the ER. The glycoprotein

thus formed is then transported to the Golgi where

removal of further mannose residues may take

place leading to a 'core' structure containing 3

mannose, and 2 N-acetylglucosamine residues, which

may then be elongated with a variety of different

monosaccharides including galactose, N-acetyl-

glucosamine, N-acetylgalactosamine, fucose and sialic

acid.

N-linked glycosylation (CTM+PTM)

Asn-X-Ser, Asn-X-Thr or Asn-X-Cys, where X could be any amino acid except Pro

CHALLENGE: complex, diversified structures; no chromophore /

fluorophore groups; mostly not charged

Analytical methods in glycan analysis:

• Gas Chromatography

• Structural characterization options: MS and NMR

• PAGE

• HPLC: - HPAE/PAD

- Normal phase and HILIC (HPLC and UPLC)

- Graphitized carbon (HPLC and chipLC)

• Capillary Electrophoresis / Microfluidics

Glycan analysis options

GLYCOPROTEINS Sample preparation for CGE based analysis

1. Release of N-linked glycan structures by

Peptide N-glycosidase F (PNGaseF)

digestion

2. Removal of the deglycosylated proteins by

ice-cold ethanol precipitation

3. Labeling of the released sugar structures by

reductive amination using l-aminopyrene-

3,6,8-trisulfonic acid (APTS)

APTS labeling reaction of carbohydrates

Purpose:

Introduction of label and charge

• Reductive amination

• Sugar reducing ends only

• ex 450 - 490 / em 520 nm LIF, excellent sensitivity

• Simple, one step reaction

• Great efficiency (over 90%) under optimized conditions

(reagent concentration, time, temperature, pH, solvent)

• Non-selective: uniform labeling for most structures

• Easy quantification: one fluorophore per sugar structure

Sample purification options for excess APTS

removal

1) Size exclusion chromatography using 96 well filter

plate filled with 100 ml Sephadex G10 resin

2) G10 bead filled pipette tips

- 200 ul pipette tips filled with 160 ul G10 resin

- conditioning and elution with 50 % acetonitrile

3) DPA-6S bead filled pipette tips

- 1000 ul pipette tips filled with 10 ul DPA-6S

normal phase polyamide resin

- washing: 95% acetonitrile

- elution: 20% acetonitrile

Sample purification results

High Throughput Glycan Analysis of purified

samples

Glycan analysis of various glycoproteins

R e l

a t i v

e F

l u o r e

s c e n

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n i t s

5.0

10.0

15.0

1.0

6.0

Time (min) Time (min)

10.0 15.0 20.0 25.0 30.0 20.0

1.0

6.0

R e l

a t i v

e F

l u o r e

s c e n

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n i t s

A B

Fetuin

Ribonuclease B

F1

F2 F3

F4

F3, F4

F1, F2

M5

M6

M7 a,b,c

M8 a,b,c

M9

M5

M6

M7 a,b,c

M8 a,b,c

M M9

12 cm capillary 30 cm capillary

Alpha-1-acid glycoprotein

Fetuin

Ribonuclease B

IgG

G2 G1

G0

Human plasma sample preparation

Removal of the relatively high blood-sugar (glucose) content

form human plasma samples prior to CGE based glycan analysis

C18 chromatographic stationary phase

filled pipette tips

Ultrafiltration with:

- 3 kDa sieving

- 10 kDa sieving

CGE profiling of human plasma samples

Without glucose removal After glucose removal

5.0

10.0

15.0

20.0

10.0 20.0 30.0

15.0 20.0

10.0 20.0 30.0

5.0

10.0

15.0

20.0

15.0 20.0 25.0

R e l

a t i v

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l u o r e

s c e n

c e U

n i t s

R e l

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s c e n

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Time (min) Time (min)

1.0

4.0

8.0 1.0

0.6

0.2

A B

APTS GLU APTS

GLU

Boronic acid – Lectin Affinity Chromatography

(BLAC) enrichment of glycoproteins

Lectin - Sugar Specificities

AAL: Aleuria Aurantia Lectin; LTL: Lotus Tetragonolobus Lectin; UEA I: Ulex Europaeus Agglutinin I; ACL: Amaranthus

Caudatus Lectin; ECL: Erythrina Cristagalli Lectin; EEL: Erythrina Cristagalli Lectin; GSL I: Griffonia (Bandeiraea)

Simplicifolia Lectin I; MAL I: Maackia Amurensis Lectin I; PNA: Peanut Agglutinin; RCA I: Ricinus Communis Agglutinin

I; RCA II: Ricinus Communis Agglutinin II; SBA: Soybean Agglutinin; ConA: Concanavalin A; LCA: Lens Culinaris

Agglutinin; PSA: Pisum Sativum Agglutinin; GNL: Galanthus Nivalis Lectin; HHL: Hippeastrum Hybrid Lectin; NPL:

Narcissus Pseudonarcissus Lectin; BPL: Bauhinia Purpurea Lectin; DBA: Dolichos Biflorus Agglutinin; MPL: Maclura

Pomifera Lectin; PTL: Psophocarpus Tetragonolobus Lectin; SJA: Sophora Japonica Agglutinin; VVA: Vicia Villosa

Lectin; WFA: Wisteria Floribunda Lectin; DSL: Datura Stramonium Lectin; GSL II: Griffonia (Bandeiraea) Simplicifolia

Lectin II; LEL: Lycopersicon Esculentum (Tomato) Lectin; STL: Solanum Tuberosum (Potato) Lectin; WGA: Wheat

Germ Agglutinin; MAL II: Maackia Amurensis Lectin II; SNA: Sambucus Nigra Lectin; PHA-E: Phaseolus vulgaris

Erythroagglutinin; PHA-L: Phaseolus vulgaris Leucoagglutinin

Boronic Acid Complexation of Glycoproteins

Binding properties of various proteins to wheat germ

agglutinin, concanavalin A and boronic acid

Ribonuclease B affinity to concanavalin (Con A)

bound agarose beads

(a) as a function of binding buffer pH (b) as a function of NaCl concentration

SDS-PAGE analysis of BLAC (Con A)(left) and BLAC

(WGA)(right) column elution fractions

1) Protein molecular mass standard; 2) myoglobin; 3)

ribonuclease B; 4) glutathione peroxidase (GPO); 5)

elution by boronic acid elution buffer (GPO+RNaseB); 6)

elution by Con A elution buffer (RNaseB).

1) protein molecular mass standard; 2) myoglobin; 3)

ribonuclease B; 4) trypsin inhibitor (TI); 5) elution by

boronic acid elution buffer (RNaseB); 6) elution by

WGA elution buffer (TI).

BLAC (Con A) BLAC (WGA)

CE traces of selective (upper trace) and combined

(lower trace) BLAC (WGA) column elution fractions

Upper trace: Injection of the mixture of ribonuclease B (1) and trypsin inhibitor (2) followed by

elution using the boronic acid elution buffer (selective elution).

Lower trace: Injection of the same mixture followed by elution using the combined boronic acid

+ WGA elution buffer (combined elution).

BLAC enrichment of trypsin inhibitor

(chicken egg white, ovomucoid)

Upper panel: RP-HPLC analysis of trypsin inhibitor (Sigma, high lysozyme content)

Fractions collected: 1A, 1B and 2 (the insets show thei MALDI spectra of 1A+B and 2)

Lower panel: BLAC partitioning of 1A, 1B and 2 fractions

1 2

RP-HPLC analysis after glycoaffinity partitioning of

the model protein mixture at different temperatures

Human serum glycan profiling after boronic acid,

Con A and BLAC affinity resin purification

Boronic Acid

BLAC / Con A

Con A

Profiling of normal human plasma glycans with (A)

and w/o (B) BLAC enrichment

Malignant cells release glycoproteins carrying disease-related

glycans into the interstitial space

Drake, P. M. et al. Clin Chem 2010;56:223-236

The glycoprotein products of tumor cells carry aberrant carbohydrate structures

compared to their normal counterparts. Typical changes include increased levels of

fucose (red triangle) and sialic acid (purple diamond), the addition of

polylactosamine units [repeating sequences of galactose (yellow circle) and N-

acetylglucosamine (blue square)], and higher-order branching of N-linked glycans.

Chapter 44, Figure 1 Essentials of Glycobiology

Second Edition

The increased size of N-glycans that occurs upon malignant transformation

can be explained by an elevation in GlcNAc transferase-V (GNT-V) activity

Human Serum Glycome Profiling by CE

N-linked glycan profiling of pooled healthy and prostate

cancer patient sera after BLAC partitioning

Fuc

Summary

ACKNOWLEDGMENT

Günther Bonn

FP 6 MXC and STREP grants from the European Commission

GenAu national and OAD international grants

PhyNexus Inc, San Jose (CA)

Marcell Olajos

Viktoria Vukics

Lorenzo De Benedictis

Zuly Rivera-Monroy

Alex Monzo-Fuentes

Javier Otero

Stefan Mittermayr

Agnes Szilagyi

Eszter Szantai

In Memoriam Professor Csaba Horváth

1930 - 2004

International Recognition

1978 Dal Nogare Award

1978 Commemorative Tswett Medal (USSR)

1980 M.S. Tswett Award in Chromatography

1982 Humboldt Award for Senior US Scientists

1983 American Chemical Society National

Chromatography Award

1986 Chromatography Award of the Eastern

Analytical Symposium

1990 Member of the Hungarian Academy of Science

1994 A.J.P. Martin Gold Medal

1994 Fellow of the AIChE

1997 Halász Medal Award

2000 Michael Widmer Award of the New Swiss

Chemical Society

2001 American Chemical Society National Award

2002 Cross of Honor for Arts and Sciences of the

Austrian Republic

2003 Torbern Bergman Medal of the Swedish

Chemical Society

2003 Heureka Price of the Hungarian Chemical

Society

2004 Member of the US National Academy of

Engineering

1952 M.S., Technical University Budapest (BME)

1952-56 Faculty member at BME

1957-61 Farbwerke Hoest

1961-63 Ph.D. at J.W.Goethe University, Frankfurt

1963-64 Harvard Medical School

1964-70 Yale University

1970-79 Associate Professor, Dept. Eng. Yale

1979-2004 Professor of Chemical Engineering, Yale

Llewelyn West Jones Jr. Professor of Chem. Eng. 1993-1998

Roberto C. Goizueta Professor of Chem. Eng. 1998-2004