Online citations, reference lists, and bibliographies.
← Back to Search

Sialic Acid Methylation Refines Capillary Electrophoresis Laser‐induced Fluorescence Analyses Of Immunoglobulin G N‐glycans Of Ovarian Cancer Patients

C. Schwedler, M. Kaup, Dominique Petzold, B. Hoppe, E. I. Braicu, J. Sehouli, M. Ehlers, M. Berger, R. Tauber, V. Blanchard
Published 2014 · Chemistry, Medicine

Save to my Library
Download PDF
Analyze on Scholarcy
Alterations in IgG N‐glycosylation coincide with the development of a number of diseases including cancer and could potentially be used as diagnostic markers. CE‐LIF of 8‐aminopyrene‐1,3,6‐trisulfonic acid labeled N‐glycans is a well‐established rapid method to characterize IgG N‐glycans that needs only low amounts of starting material. However, sialylated N‐glycans have short migration times due to their negative charge. As a result, some of them are not well resolved and co‐migrate with neutral glycans. In this work, we neutralized the negative charge of sialic acids by methylation and optimized the protocol using the commercially available disialylated biantennary oligosaccharide (A2G2S2). IgG N‐glycans isolated from healthy human serum were then analyzed using this method. We could demonstrate that co‐migration of A2, FA2G2S1, and FA2B[3]G1S1 was prevented, which allowed an accurate quantification of these N‐glycans. Finally, we investigated the IgG N‐glycan profiles of patients suffering from ovarian cancer using the conventional and methylation methods. With both methods, we observed an increase of agalactosylated structures that was accompanied by a decrease in digalactosylated structures. Finally, using the methylation protocol, we could further demonstrate an increase of A2, which was technically impossible with the conventional method.
This paper references
Oligosaccharide sequencing technology
P. Rudd (1997)
Intracellular functions of N-linked glycans.
A. Helenius (2001)
Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography.
B. Kuester (1997)
Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.
R. Saldova (2007)
Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis.
S. Kamoda (2004)
Biomarker discovery for inflammatory bowel disease, using proteomic serum profiling.
Marie-Alice Meuwis (2007)
The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity*
T. Shinkawa (2003)
Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris
Véronique Blanchard (2006)
Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity
F. Nimmerjahn (2007)
The effect of individual N-glycans on enzyme activity.
D. Skropeta (2009)
Analysis of the Oligosaccharide Chain of Human Serum Immunoglobulin G in Patients with Localized or Metastatic Cancer
Y. Kanoh (2004)
A Serum Glycomics Approach to Breast Cancer Biomarkers*S
C. Kirmiz (2007)
N-linked glycan structures and their expressions change in the blood sera of ovarian cancer patients.
W. Alley (2012)
Glycans: bioactive signals decoded by lectins.
H. Gabius (2008)
Mass spectrometry in the analysis of N-linked and O-linked glycans.
S. J. North (2009)
Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin affinity column.
Ziping Yang (2004)
Glycosylation of human IgG-Fc: influences on structure revealed by differential scanning micro-calorimetry.
R. Ghirlando (1999)
Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS
Z. Szabó (2010)
Chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated N-linked glycans: a potential methodology for cancer-biomarker discovery.
W. Alley (2010)
Monosaccharide analysis of glycoconjugates by high-performance anion-exchange chromatography with pulsed amperometric detection.
M. Hardy (1989)
Structure-based engineering of a monoclonal antibody for improved solubility.
Sheng-Jiun Wu (2010)
Chemical glycobiology : Carbohydrates and glycobiology
C. Bertozzi (2001)
Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.
J. Bones (2010)
The impact of glycosylation on the biological function and structure of human immunoglobulins.
J. N. Arnold (2007)
A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles.
G. R. Guile (1996)
Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications.
X. Liu (2008)
Stabilization of sialic acids in N-linked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption/ionization mass spectrometry.
A. Powell (1996)
The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: properties of a series of truncated glycoforms.
Y. Mimura (2000)
High-throughput solid-phase permethylation of glycans prior to mass spectrometry.
P. Kang (2008)
Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies.
S. Kamoda (2006)
The N-linked sugar chains of human immunoglobulin G: their unique pattern, and their functional roles.
A. Kobata (2008)
High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid.
K. Anumula (1998)
Human IgG Fc-glycosylation profiling reveals associations with age, sex, female sex hormones and thyroid cancer.
G. Chen (2012)
Multiplexed analytical glycomics: rapid and confident IgG N-glycan structural elucidation.
S. Mittermayr (2011)
High Throughput Isolation and Glycosylation Analysis of IgG–Variability and Heritability of the IgG Glycome in Three Isolated Human Populations*
M. Pučić (2011)
High throughput plasma N-glycome profiling using multiplexed labelling and UPLC with fluorescence detection.
A. Knežević (2011)
Mass Spectrom
A K Powell (1996)
Total serum protein N‐glycome profiling on a capillary electrophoresis‐microfluidics platform
N. Callewaert (2004)
"One-pot" methylation in glycomics application: esterification of sialic acids and permanent charge construction.
X. Liu (2007)
Fcγ receptors as regulators of immune responses
F. Nimmerjahn (2008)
Rapid analysis of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) by reversed-phase ultra-performance liquid chromatography with fluorescence detection and electrospray ionization time-of-flight mass spectrometry.
Takamasa Kurihara (2009)
The carbohydrate at FcgammaRIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms.
Claudia Ferrara (2006)
Control of Recombinant Monoclonal Antibody Effector Functions by Fc N‐Glycan Remodeling in Vitro
Jason Hodoniczky (2005)
Glycans in cancer and inflammation — potential for therapeutics and diagnostics
Danielle H Dube (2005)
Comparison of cell lines for stable production of fucose‐negative antibodies with enhanced ADCC
Yutaka Kanda (2006)
Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality.
Bernard J Scallon (2007)
Effect of posttranslational modifications on the thermal stability of a recombinant monoclonal antibody.
Hongcheng Liu (2006)
Immunoglobulin G Fc N-glycan profiling in patients with gastric cancer by LC-ESI-MS: relation to tumor progression and survival
K. Kodar (2011)
High-resolution carbohydrate profiling by capillary gel electrophoresis
A. Guttman (1996)
Endo-β-N-acetylglucosaminidase H de-N-glycosylation in a domestic microwave oven: application to biomarker discovery.
E. Frisch (2013)
Rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry.
Yoshiaki Miura (2007)

This paper is referenced by
Recent advances in capillary electrophoresis separation of monosaccharides, oligosaccharides, and polysaccharides
V. Mantovani (2018)
Filariasis asymptomatically infected donors have lower levels of disialylated IgG compared to endemic normals
N. L. O’Regan (2014)
The Anterior Spreader Flap: A Minimally Invasive Alternative to the Auto Spreader Flap in the Treatment of Patients with Nasal Valve Dysfunction
S. Bessler (2014)
Expanding the Reaction Space of Linkage-Specific Sialic Acid Derivatization
Tamas Pongracz (2019)
Enhanced detection of in‐gel released N‐glycans by MALDI‐TOF‐MS
S. Weiz (2015)
Measurement of Neutral and Sialylated IgG N-Glycome at Asn-297 by CE-LIF to Assess Hypogalactosylation in Rheumatoid Arthritis.
C. Schwedler (2019)
Acute‐phase glycoprotein N‐glycome of ovarian cancer patients analyzed by CE‐LIF
S. Weiz (2016)
Immunoglobulin G Subclass-Specific Glycosylation Changes in Primary Epithelial Ovarian Cancer
M. Wieczorek (2020)
Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery
C. Schwedler (2014)
Structural Characterization of Serum N-Glycans by Methylamidation, Fluorescent Labeling, and Analysis by Microchip Electrophoresis.
I. Mitra (2016)
Comparison of 2-Aminobenzamide, Procainamide and RapiFluor-MS as Derivatizing Agents for High-Throughput HILIC-UPLC-FLR-MS N-glycan Analysis
Toma Keser (2018)
Serum Glycans as Risk Markers for Non–Small Cell Lung Cancer
L. Ruhaak (2016)
Hypogalactosylation of immunoglobulin G in rheumatoid arthritis: relationship to HLA-DRB1 shared epitope, anticitrullinated protein antibodies, rheumatoid factor, and correlation with inflammatory activity
C. Schwedler (2018)
Capillary Electrophoresis Separations of Glycans
Grace Lu (2018)
Semantic Scholar Logo Some data provided by SemanticScholar