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Proteomic Profiling pp 355–368Cite as

Enrichment and Identification of Bacterial Glycopeptides by Mass Spectrometry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1295))

Abstract

Large-scale analysis of protein N- and O-linked glycosylation by mass spectrometry has traditionally been performed in eukaryotes by parallel approaches aimed at elucidating glycan structures (glycomics) and their formerly glycosylated peptides (glycoproteomics) without reference to their intact state. Such analyses depend heavily on commercial glycosidases (e.g. protein N-glycosidase F) that can remove glycans from the peptide backbone for separate analyses. Bacterial glycosylation has only recently been identified as a widespread phenomenon. In many cases however, unique bacterial sugars preclude enzymatic removal, therefore ultimately requiring a site-specific approach for intact glycopeptide analysis. Here, we describe protocols for the enrichment of bacterial glycopeptides using zwitterionic–hydrophilic interaction liquid chromatography (ZIC-HILIC) and their analysis using liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) with collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) fragmentation for glycan structure elucidation and glycopeptide identification.

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Acknowledgments

This work was supported by an Australian Research Council Discovery Project Grant to S.J.C. (ARC DP 110103573). N.E.S. is supported by a National Health and Medical Research Council of Australia (NHMRC) Overseas (Biomedical) Early Career Fellowship (APP1037373) and a Michael Smith Foundation for Health Research Trainee Postdoctoral Fellowship (award # 5363).

Author information

Authors and Affiliations

  1. School of Molecular Bioscience, The University of Sydney, Sydney, NSW, 2006, Australia

    Nichollas E. Scott & Stuart J. Cordwell

  2. Centre for High-Throughput Biology, Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, BC, Canada, V6T 1Z4

    Nichollas E. Scott

  3. Discipline of Pathology, School of Medical Sciences, The University of Sydney, Sydney, NSW, 2006, Australia

    Stuart J. Cordwell

  4. Charles Perkins Centre, The University of Sydney, The Hub Building D17, Sydney, NSW, 2006, Australia

    Stuart J. Cordwell

Authors
  1. Nichollas E. Scott
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  2. Stuart J. Cordwell
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Correspondence to Stuart J. Cordwell .

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Editors and Affiliations

  1. Bio-Rad Laboratories GmbH, Munich, Germany

    Anton Posch

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Scott, N.E., Cordwell, S.J. (2015). Enrichment and Identification of Bacterial Glycopeptides by Mass Spectrometry. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_25

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  • DOI: https://doi.org/10.1007/978-1-4939-2550-6_25

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2549-0

  • Online ISBN: 978-1-4939-2550-6

  • eBook Packages: Springer Protocols

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