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Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery

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Abstract

Alterations in glycosylation have been observed in many human diseases and specific changes in glycosylation have been proposed as relevant diagnostic information. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is a robust method to quantify desialylated N-glycans that are labeled with 8-aminopyrene-1,3,6-trisulfonic acid prior to analysis. To date, only a maximum of 12 glycan structures, the most abundant ones, have been identified by CE-LIF to characterize glycome modulations of total serum in the course of the diseases. In most forms of cancer, findings using CE-LIF were limited to the increase of triantennary structures carrying a Lewisx epitope. In this work, we identified 32 linkage and positional glycan isomers in healthy human serum using exoglycosidase digestions as well as standard glycoproteins, for which we report the assignment of novel structures. It was possible to identify and quantify 34 glycan isomers in the serum of primary epithelial ovarian cancer patients (EOC). Reduced levels of diantennary structures and of high-mannose 5 were statistically significant in the EOC samples, and also, elevated branching as well as increased antennary fucosylation were observed. For the first time, we could demonstrate that not only antennary fucosylation was of relevance in tetraantennary structures but also core-fucosylated tetraantennary N-glycans were statistically increased in EOC patients. The results of the current study provide an improved dataset to be used in glycan biomarker discovery.

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Abbreviations

ABS:

Recombinant neuraminidase III from Arthrobacter ureafaciens

ACN:

Acetonitrile

AGP:

α1-Acid glycoprotein

AMF:

Almond meal α-fucosidase

APTS:

8-Aminopyrene-1,3,6-trisulfonic acid

BKF:

Bovine kidney α-fucosidase

CE-LIF:

Capillary electrophoresis coupled with laser-induced fluorescence

EOC:

Epithelial ovarian cancer

GU:

Glucose units

IgG:

Immunoglobulin G

JBH:

ß-N-Acetylhexosaminidase from jack bean

MALDI-TOF-MS:

Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry

PNGase F:

Peptide-N4-(acetyl-ß-glucosaminyl) asparagine amidase F

RNase B:

Ribonuclease B

SPG:

Recombinant ß-galactosidase from Streptococcus pneumonia

SPH:

ß-N-Acetylhexosaminidase cloned from Streptococcus pneumonia expressed in E. coli

TFA:

Trifluoroacetic acid

THF:

Tetrahydrofuran

XMG:

Recombinant ß-galactosidase II from Xanthomanas manihotis

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Acknowledgments

This work was supported by the German Federal Ministry of Education and Research (BMBF) through InnoProfile (grant 03IP511) and ArthroMark (grant 01EC1009A), by the foundation of Pathobiochemistry and Molecular Diagnostics, and by the Sonnenfeld foundation.

Conflict of interest

The authors declare no conflicts of interest.

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Authors and Affiliations

  1. Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité Medical University, Augustenburger Platz 1, 13353, Berlin, Germany

    Christian Schwedler, Matthias Kaup, Stefan Weiz, Maria Hoppe, Berthold Hoppe, Rudolf Tauber, Markus Berger & Véronique Blanchard

  2. Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Takustrasse 3, 14195, Berlin, Germany

    Christian Schwedler

  3. Laboratory Medicine and Toxicology, Labor Berlin–Charité Vivantes GmbH, Sylter Straße 2, 13353, Berlin, Germany

    Berthold Hoppe

  4. Department of Gynecology, Charité Medical University, Augustenburger Platz 1, 13353, Berlin, Germany

    Elena Iona Braicu & Jalid Sehouli

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  1. Christian Schwedler
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  2. Matthias Kaup
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  3. Stefan Weiz
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  4. Maria Hoppe
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  10. Véronique Blanchard
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Corresponding author

Correspondence to Véronique Blanchard.

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Schwedler, C., Kaup, M., Weiz, S. et al. Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery. Anal Bioanal Chem 406, 7185–7193 (2014). https://doi.org/10.1007/s00216-014-8168-y

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  • DOI: https://doi.org/10.1007/s00216-014-8168-y

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