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Sialic Acid Capture-and-Release and LC-MSn Analysis of Glycopeptides

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Mass Spectrometry of Glycoproteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 951))

Abstract

Extracellular glycoproteins frequently carry terminal sialic acids on their N-linked and/or O-linked glycan structures. In this chapter a sialic acid specific capture-and-release protocol for the enrichment of N- and O-glycopeptides originating from glycoproteins in complex biological samples is described. The enriched glycopeptides are subjected to reversed phase liquid chromatography (LC) interfaced with electrospray ionization and multistage tandem mass spectrometry (MSn). The glycopeptide precursor ions are fragmented by collision-induced dissociation (CID) for analysis of the glycan parts in the MS2 spectra. Further fragmentation (i.e., MS3) of deglycosylated peptide ions results in peptide backbone fragmentation, which is used in protein database searches to identify protein sequences. For O-glycopeptides the use of both CID and electron capture dissociation (ECD) fragmentation of the peptide backbone with intact glycans still attached are used to pinpoint the glycosylation sites of glycopeptides containing several Ser/Thr residues. The step-by-step protocols for fragmentation analyses of O- and N-glycopeptides enriched from human cerebrospinal fluid are described.

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Acknowledgment

We thank Adnan Halim, Ammi Grahn, Ulla RĂĽetschi, Gunnar Brinkmalm, Henrik Zetterberg, and Kaj Blennow for individual contributions to the glycoproteomic project. Funding from the Swedish Medical Research Council (8266) and governmental ALF grants to the Sahlgrenska University Hospital are gratefully acknowledged.

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Authors and Affiliations

  1. Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, the Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden

    Jonas Nilsson & Göran Larson

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  1. Jonas Nilsson
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  2. Göran Larson
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Correspondence to Jonas Nilsson .

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Editors and Affiliations

  1. Medical Center, Department of Biochemistry, University of Texas Southwestern, Harry Hines Blvd. 5323, Dallas, 75390-9038, Texas, USA

    Jennifer J. Kohler

  2. Medical Center, Dallas, University of Texas Southwestern, Harry Hines Blvd. 5323, Dallas, 75390-9185, Texas, USA

    Steven M. Patrie

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Nilsson, J., Larson, G. (2013). Sialic Acid Capture-and-Release and LC-MSn Analysis of Glycopeptides. In: Kohler, J., Patrie, S. (eds) Mass Spectrometry of Glycoproteins. Methods in Molecular Biology, vol 951. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-146-2_7

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  • DOI: https://doi.org/10.1007/978-1-62703-146-2_7

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-145-5

  • Online ISBN: 978-1-62703-146-2

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