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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
c e U
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
c e U
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
e F
l u o r e
s c e n
c e U
n i t s
R e l
a t i v
e F
l u o r e
s c e n
c e U
n i t s
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