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Site-Specific N- and O-Glycopeptide Analysis Using an Integrated C18-PGC-LC-ESI-QTOF-MS/MS Approach

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High-Throughput Glycomics and Glycoproteomics

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

Abstract

The vast heterogeneity of protein glycosylation, even of a single glycoprotein with only one glycosylation site, can give rise to a set of macromolecules with different physicochemical properties. Thus, the use of orthogonal approaches for comprehensive characterization of glycoproteins is a key requirement. This chapter describes a universal workflow for site-specific N- and O-glycopeptide analysis. In a first step glycoproteins are treated with Pronase to generate glycopeptides containing small peptide sequences for enhanced glycosylation site assignment and characterization. These glycopeptides are then separated and detected using an integrated C18-porous graphitized carbon-liquid chromatography (PGC-LC) setup online coupled to a high-resolution electrospray ionization (ESI)-quadrupole time-of-flight (QTOF)-mass spectrometer operated in a combined higher- and lower-energy CID (stepping-energy CID) mode. The LC-setup allows retention of more hydrophobic glycopeptides on C18 followed by subsequent capturing of C18-unbound (glyco)peptides by a downstream placed PGC stationary phase. Glycopeptides eluted from both columns are then analyzed within a single analysis in a combined data acquisition mode. Stepping-energy CID results in B- and Y-ion fragments originating from the glycan moiety as well as b- and y-ions derived from the peptide part. This allows simultaneous site-specific identification of the glycan and peptide sequence of a glycoprotein.

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Author information

Authors and Affiliations

  1. Division of BioAnalytical Chemistry, VU University Amsterdam, De Boelelaan 1083, Amsterdam, 1081, HV, The Netherlands

    Kathrin Stavenhagen & Manfred Wuhrer

  2. Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, Leiden, 2333, ZA, The Netherlands

    Kathrin Stavenhagen & Manfred Wuhrer

  3. Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, 14424, Germany

    Hannes Hinneburg

  4. Department of Biology, Chemistry, Pharmacy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, 14195, Germany

    Hannes Hinneburg

  5. Department of Biomolecular Systems, Max Planck Institute of Colloids andInterfaces, Am Mühlenberg 1, Potsdam, 14476, Germany

    Daniel Kolarich

Authors
  1. Kathrin Stavenhagen
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  2. Hannes Hinneburg
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  3. Daniel Kolarich
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  4. Manfred Wuhrer
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Corresponding author

Correspondence to Manfred Wuhrer .

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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Stavenhagen, K., Hinneburg, H., Kolarich, D., Wuhrer, M. (2017). Site-Specific N- and O-Glycopeptide Analysis Using an Integrated C18-PGC-LC-ESI-QTOF-MS/MS Approach. 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_9

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  • DOI: https://doi.org/10.1007/978-1-4939-6493-2_9

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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

  • eBook Packages: Springer Protocols

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