CAPILLARY ELECTROPHORESIS OF GLYCANS

Biotechnology and biomedical applications

András Guttman

The Barnett Institute, Northeastern University, Boston, MAand

Horváth Laboratory of Bioseparation Sciences, Debrecen, Hungary

MASSEP, Boson, MA, Dec 21, 2010

Significance of protein glycosylation

There has been a rapid recent progress in glycomicsresearch to understand the biological role ofcarbohydrate moieties of glycoproteins.Alterations in protein glycosylation with variablesite occupancy or changes in oligosaccharide structures(microheterogeneity) can lead to biological activitychanges. Glycosylation types: N-linked glycosylation(through the amide nitrogen of asparagine side chains)and O-linked glycosylation (through the hydroxyoxygen of serine and threonine side chains).Electron micrograph of the glycocalyx

at the surface of an erythrocyte.

For N-linked oligosaccharides, a 14-sugar precursor is first added to theasparagine in the polypeptide chain ofthe target protein. The structure ofthis precursor is common to mosteukaryotes, and contains 3 glucose, 9mannose, and 2 N-acetylglucosaminemolecules. A complex set of reactionsattaches this branched chain to acarrier molecule (dolichol), and then itis transferred to the appropriate pointon the polypeptide chain as it istranslocated into the ER lumen.

N-linked glycosylation

Types of N-linked glycosylation: - High-mannose type: contains just two N-acetylglucosamines with many

mannose residues.- Complex type: can contain almost any number of the other types of

sugars, including more than the original two N-acetylglucosamines.- Hybrid type: combination of the two above.

Chapter 44, Figure 1Essentials of Glycobiology

Second Edition

The increased size of N-glycans that occurs upon malignant transformationcan be explained by an elevation in GlcNAc transferase-V (GNT-V) activity

Malignant cells release glycoproteins carrying disease-related glycans into the interstitial space, where they can reach the circulation

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

Challenge: complex, diversified structures; no chromophore / fluorophore groups; mostly not charged

Advantages of CGE over other analytical methods• GC requires derivatization resulting in stereoisomers and complex separation

patterns.• HPLC: - HPAE/PAD: limited separation efficiency, non-specific detector response

- Normal phase and HILIC: time consuming, low efficiency,- UPLC: no current informatics solution- Graphitized carbon: excessive retention of sialylated structures

• Structural characterization – MS (limited information on anomericconfiguration and other isoforms), NMR (high quantity and purityrequirements).

• PAGE – slow, labor intensive and not quantitative.• CE – fast, efficient, automated, very sensitive, small sample volume

requirements, easy multiplexing, different modes of separation; however,requires charged UV/fluorophore tags to assure electromigration and UV/LIFdetectability. Informatics database is being built.

Glycan analysis options

High separation efficiency Small sample volume (1-10 l) Fast separation (min - sec) Predictable selectivity Full automation Quantification/good dynamic range Reproducibility/robustness Multi-capillary option (CAE) Multi-spectral imaging Coupling to mass spectrometer

CAPILLARY ELECTROPHORESIS

-+

samplebuffer buffer

detector

SEPARATION MODES IN CAPILLARY ELECTROPHORESIS

Capillary zone electrophoresis (CZE) Micellar electrokinetic chromatography (MEKC) Capillary gel electrophoresis (CGE) Capillary isoelectric focusing (CIEF) Capillary affinity electrophoresis (CAE) Capillary isotachophoresis (ITP) Capillary electrochromatography (CEC) Micropreparative applications

CGE: From the first oligonucleotide separationto sequencing the Human Genome

1988 1996

A.Guttman, A.Paulus, A.S.Cohen, B.L.Karger and H.Rodriguez, W.S.Hancock, High Performance Capillary Gel Electrophoresis: High Resolution and Micropreparative Applications, Electrophoresis'88, Proceedings (Ed.: C.Schafer-Nielsen) VCH, Weinheim, Germany, (1988)

B.L.Karger and A.Guttman, From capillaries to the Genome,Gen.Prot.Techn, 3 (2003) 12-16.

A.Guttman and T.Pritchett, Capillary Gel Electrophoresis Separation of High-mannose Type Oligosaccharides derivatized by 1-aminopyrene-3,6,8-trisulfonic acid Electrophoresis, 16 (1995) 1906-1911.

10.00 12.00 14.00Time (min)

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ence

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U, x

1E+0

0)

M5

M6

M7ABC

M8ABC

M9 Rib-B

Man-5Man-6Man-7Man-8Man-9

Separation of APTS labeled high-mannose type glycans released from bovine ribonuclease B

Inset: Structural representation of the high-mannose type N-linked oligosaccharides.

46633

Man 5

466332

Man 6466332 Man 7D12

466332 Man 7D2

2

466332 Man 7D32

466332 Man 8D12

2

466332 Man 8D2

22

466332 Man 8D3

2

2

4663322

22

Man 9

raw data aligned data

Migration time normalization for CAE operation

J.Khandurina, A.A.Anderson, N.A.Olson, J.Stege, A.Guttman, Large-scale carbohydrate analysis by capillary arrayelectrophoresis. Part 2. Data normalization and quantification, Electrophoresis 25 (2004) 3122-3127.

Sample preparation for CGE based N-linked glycan analysis

1. Release of N-linked glycan structures by Peptide N-glycosidase F (PNGaseF) digestion

2. Removal of the deglycosylated proteins - ice-cold ethanol precipitation/ centrifugation- membrane filtration

3. Labeling of the released sugar structures by reductive amination using l-aminopyrene-3,6,8-trisulfonic acid (APTS)

Methods to Accelerate Enzyme Catalyzed N-deglycosylation of Glycoproteins

• Microwave assisted deglycosylation of N-linked glycans

• Immobilized PNGase F enzyme reactors in capillary

columns

• Integrated microfluidic chip for rapid deglycosylation

• Pressure cycling technology (PCT)

• Kinetic advantage: pressure promotes water dissociation• Many hydrolytic reactions are accelerated• Substrate binding – pressure reversibly denatures

substrate protein, revealing hindered cleavage sites • PCT accelerates and improves reduction/alkylation• Enzymes: Trypsin, Chymotrypsin, Pepsin, Lys-C, Glu-C,

Asp-N, Proteinase K, PNGase F tested to date – all positive

• Both in-solution and in-gel digestion protocols benefit from PCT

PCT- enhanced enzyme reactions

Comparative CE Analysis of APTS Labeled Released Glycans from Polyclonal Human IgG Using PCT and Atmospheric N-deglycosylation

with 1:2500 Enzyme:Substrate Molar Ratio.

A) APTS labeled maltooligosaccharide ladderB) PCT: 30 kPsi, 5 min. 37oC C) Atmospheric pressure, 3 hours, 37oC D) Atmospheric pressure, 5 min, 37oC IS2: maltose - APTS.

Z.Szabo, A.Guttman, B.L.Karger, Rapid release of N-linked glycans from glycoproteins by pressure cycling technology, Analytical Chemistry 82 (2010) 2588-2593.

Advantages of Pressure Cycling Technology (PCT) Assisted Enzymatic N-deglycosylation

• The high pressure facilitates conformation changes of the target glycoprotein, increasing the accessibility of the endoglycosidase to the cleavage sites.

• 1:2500 enzyme : substrate molar ratio at 30 kPsi and 37oC quantitatively released the asparagine linked glycans in minutes.

• Pressure cycling apparently did not lead to any loss of sialic acid residues.

• The microliter scale reaction volume alleviated possible precipitation related issues.

• PCT offers simultaneous processing of 12 samples.

Sugar labeling by APTS

Purpose: Introduction of label and charge

• Reductive amination

• Sugar reducing ends only

• ex 488 nm / 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 molecule

A.Guttman, A.Chen, R.Evangelista and N.Cooke, High Resolution Capillary Gel Electrophoresis of Reducing Oligosaccharides Labeled with 1-Aminopyrene-3,6,8-trisulfonate (APTS), Anal.Biochem., 233 (1996) 234-242

Trisialylated triantennary (2 x 2,3) oligosaccharide, derivatization time: 2 hours at all temperatures.

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Desialylation (%

)

Labeling

Desialylation

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yiel

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)

Temperature (oC)

75oC

Derivatization yield and desialylation kinetics

Time Dependence of APTS Derivatization of 5 nmol Maltoheptaose

Solid line: 10x excess of APTS, 0.6 M citric acid catalyst Dotted line: 100x excess of APTS, 7.5% acetic acid catalystReaction temperature: 55oC

Z.Szabo, A.Guttman, T.Rejtar, B.L.Karger, Improved sample preparation method for glycan analysis of glycoproteins by CE-LIF and CE-MS, Electrophoresis 31 (2010) 1389-1395.

Sample purification options for excess APTS removal

1) Size exclusion chromatography using 96 well filter plate filled with 100 ul 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) Normal phase bead filled pipette tips- 1000 ul pipette tips filled with 10 ul DPA-6S

normal phase polyamide resin- washing: 95% acetonitrile / 5% water- elution: 20% acetonitrile / 80% water

Sample purification results

M.Olajos, P.Hajos, G.K.Bonn, A.Guttman, Sample preparation for the analysis of complex carbohydrates bymulti-capillary gel electrophoresis with LED induced fluorescence detection, Anal.Chem, 80 (2008) 4241-4246.

A.Guttman, Analysis of Monosaccharide Composition by Capillary Electrophoresis J.Chromatogr.A, 763 (1997) 271-278.

Monosaccharide composition analysis by CE

1=AMAC (9-aminoacridone, eof marker)2=Neu5Ac-AMAC; 3=GalNAc-APTS; 4=GlcNAc-APTS; 5= Man-APTS; 6=Glc-APTS; 7=Fuc-APTS;8=Gal-APTS; 9=APTS

Flowchart of N-linked oligosaccharide sequencing

Purified Glycoprotein

PNGase F Digestion 2 hours

Free N-linked Oligosaccharides

Labeling with APTS 90 min at 55oC

Labeled Oligosaccharides

Exoglycosidase Matrix Digestion 16 hours

Sequencing Digests

Combine Digests 5 min

Capillary Gel Electrophoresis

Computerized Data Analysis 10 min

Oligosaccharide Sequence

Boronic acid – Lectin Affinity Chromatography (BLAC) enrichment of glycoproteins

A.Monzo, M.Olajos, L.De Benedictis, G.K.Bonn, A.Guttman, Boronic acid - lectin affinity chromatography (BLAC): 2. Affinity micropartitioning mediated comparative glycosylation profiling, Anal.Bioanal.Chem 392 (2008) 195-201.

Comparison of affinity micropartitioning using boronic acid (A), Con A (B) and BLAC/Con A (C) resin filled pipette tips.

Profiling of normal human serum glycans with (A) and without (B) BLAC/Con A affinity enrichment.

CGE profiling of human plasma samples

Without glucose removal After glucose removal

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M.Olajos, P.Hajos, G.K.Bonn, A.Guttman, Sample preparation for the analysis of complex carbohydrates by multi-capillary gel electrophoresis with LED induced fluorescence detection, Anal.Chem, 80 (2008) 4241-4246.

Sample preparation and analysis of human serum glycoproteins by CE (glycan) and LC-MS (protein)

MAIN APPLICATIONS

FOOD AND BEVERAGE INDUSTRY

BIOTECHNOLOGY

BIOPHARMACEUTICALS

BIOMEDICAL

A.Guttman, S.Brunet and N.Cooke, Capillary Electrophoresis Fingerprinting of Carbohydrates in the Biopharmaceutical and Food/Beverage Industry. LC.GC Magazine 14 (1996) 788-792.

Capillary electrophoresis fingerprinting of oligosaccharides in beers

Ethanol from dry grind corn milling (current process)

• Starch in corn seed is ezymatically converted to sugar and fermented to ethanol

• Saccharification / Fermentation of Fiber would increase ethanol yields by 10%

Enzyme Addition

Milling Cooking Liquefaction Saccharification Fermentation

Ethanol

DDGS• Oil• Protein• Fiber

Germ Fraction(oils)

(DDGS: distillers dried grains with solubles)

High throughput enzyme activity evaluation

3 4 5 6 7 8 9 10 11 min

RFU

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G6G5G4G3G2

G1

J.Khandurina, D.L.Blum and A.Guttman, Automated Carbohydrate Profiling by Capillary Electrophoresis: A bioindustrial approach, Electrophoresis 25 (2004) 2326-2331.

Enzyme reaction monitoring:Cellooligomer standardsCellohexaose substrate

9 different cellulases

The methylotrophic yeast Pichia pastorisis a preferred host for over-expression ofrecombinant proteins as it produces largequantities of properly folded proteins andcapable of adding both N-linked and O-linked carbohydrate moieties to secretedproteins.

Over-expression of recombinant proteins in Yeast

A.Koller, J.Khandurina, J.Li, J.Kreps and A.Guttman, Structure Analysis of High-Mannose Type Oligosaccharidesby uLC/MS and Capillary Electrophoresis, Electrophoresis, 25 (2004) 2003-2009.

Phospholipase C (PLC) belongs to a class of enzymes that cleave phospholipids to produce diacylglycerol and a phosphorylated head group (e.g., inositol phosphate in the

case of phosphoinositol-specific PLCs).

MW(kDa)

A B C

30

25

20

70605040*

SDS-PAGE analysis of intact and deglycosylated PLC

(PLC band was also identified by western blotting)

1

2

After SDS-PAGE, the bands corresponding to recombinant PLC were in-gel digested withchymotrypsin.

The resulting peptides were loaded onto a 10 cm C18 RP 100 μm i.d. capillary column andeluted into a QTOF mass spectrometer by means of a 45 minute gradient.

Man

14G

lcN

Ac 2

**11

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1091

.11

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13G

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Ac 2

Man

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lcN

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Man

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lcN

Ac 2

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10G

lcN

Ac 2

Man

9Glc

NA

c 2

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8Glc

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c 2

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7Glc

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Glycopeptide analysis from recombinant PLC by μLC-MS

466

33

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Man7GlcNAc2

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Man14GlcNAc2

Digested PLC

Digested RNAse B

Undigested (control) RNAse B

Time (min)7 8 9 10 11 12 13 14 15

RFU

G3

G7

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G9G11

G13 G15

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M7 M8

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6

CE separation of high mannose type oligosccharides48 cm capillary column, I.D.: 50m; LIF detection: Ex: 488nm, Em: 520 nm. Buffer: 25 mM acetate, pH 5; temp: 25oC; Pressure injection: 10 sec at 0.5 psi.

Characterization of mAb N-linked Glycans

• IgG1 contains N-linked glycanstructures (1 conserved Asn site).

• Variability depending on expressionconditions

• Structural diversity, e.g. presence ofgalactose, sialic acid or fucose.

• Glycosylation pattern determinesBiological activity: physico-chemicalproperties, cell-mediated effectorfunctions (complement binding,activation, etc).

• Understanding the microheterogeneityof glycosylation is very important inclone selection and manufacturing oftherapeutic recombinant mAbs.

Minutes4.6 5.0 5.4 5.8 6.2 6.6 7.0 7.4

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Man 5 Man 6 Man 9

G0- F

G0 G2

G2- F

G1 G1'

G1'- FG1- F

A2

CE-MS extracted ion electropherograms of APTS-labeled sialylated biantennary glycans (panel A) and the MS traces of the corresponding major peaks (panel B)

CE – MS verification of the separated components

Z.Szabo, A.Guttman, T.Rejtar, B.L.Karger, Improved sample preparation method for glycan analysis of glycoproteins by CE-LIF and CE-MS, Electrophoresis 31 (2010) 1389-1395.

N-linked glycan profiling of pooled healthy and prostate cancer patient sera after BLAC partitioning

Fuc

F1=tetrasialo-triantennary-2x2,6; F2=tetrasialo-triantennary-2x2,3; F3=trisialo-triantennary-2x2,6; F4=trisialo-triantennary-2x2,3; M5-M9: Mannose 5 - Mannose 9.

A.Guttman, Carbohydrate Profiling by Capillary Gel Electrophoresis, Nature, 380 (1996) 461-462.

The power of CGE in N-linked glycan profiling

The future of CE and CE-MS based carbohydrate analysis

?

Barnett Institute, Boston, MA• Barry L. Karger • Zoltan Szabo • Tomas Rejtar

University of Debrecen, Hungary• Stefan Mittermayr• Akos Szekrenyes• Andras Kovacs• Janos Kerekgyarto • Csaba Varadi

NIBRT, Dublin, Ireland• Pauline Rudd• Jonathan Bones

ACKNOWLEDGMENT