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High-Throughput Glycomics and Glycoproteomics pp 97–108Cite as

High-Throughput Analysis of the Plasma N-Glycome by UHPLC

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1503))

Abstract

The understanding of glycosylation alterations in health and disease has evolved significantly and glycans are considered to be relevant biomarker candidates. High-throughput analytical technologies capable of generating high-quality, large-scale glycoprofiling data are in high demand. Here, we describe an automated sample preparation workflow and analysis of N-linked glycans from plasma samples using hydrophilic interaction liquid chromatography with fluorescence detection on an ultrahigh-performance liquid chromatography (UHPLC) instrument. Samples are prepared in 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labeling, and post-labeling cleanup with solid-phase extraction. The development of a novel approach for plasma N-glycan analysis and its implementation on a robotic platform significantly reduces the time required for sample preparation and minimizes technical variation. It is anticipated that the developed method will contribute to expanding high-throughput capabilities to analyze protein glycosylation.

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Acknowledgements

The authors acknowledge support from the European Union FP7 GastricGlycoExplorer ITN under grant agreement no. 316929. and HighGlycan under grant agreement no. 278535. We would like to acknowledge Dr. Karol Polom, Dr. Giovanni Corso, and Dr. Franco Roviello from University Hospital of Siena for providing gastric cancer serum samples. The UHPLC instrument was obtained with a grant from the IngaBritt and Arne Lundberg’s Research Foundation.

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Author notes

  1. Henning Stöckmann

    Present address: AbbVie Inc., North Chicago, IL, USA

Authors and Affiliations

  1. Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Box 440, 405 30, Gothenburg, Sweden

    Barbara Adamczyk & Niclas G. Karlsson

  2. NIBRT GlycoScience Group, NIBRT–The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co., Dublin, Ireland

    Barbara Adamczyk, Henning Stöckmann, Róisín O’Flaherty & Pauline M. Rudd

Authors
  1. Barbara Adamczyk
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  2. Henning Stöckmann
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  3. Róisín O’Flaherty
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  4. Niclas G. Karlsson
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  5. Pauline M. Rudd
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Corresponding author

Correspondence to Pauline M. Rudd .

Editor information

Editors and Affiliations

  1. Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

    Gordan Lauc

  2. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

    Manfred Wuhrer

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Adamczyk, B., Stöckmann, H., O’Flaherty, R., Karlsson, N.G., Rudd, P.M. (2017). High-Throughput Analysis of the Plasma N-Glycome by UHPLC. In: Lauc, G., Wuhrer, M. (eds) High-Throughput Glycomics and Glycoproteomics. Methods in Molecular Biology, vol 1503. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6493-2_8

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  • DOI: https://doi.org/10.1007/978-1-4939-6493-2_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6491-8

  • Online ISBN: 978-1-4939-6493-2

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