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Sialic acid capture-and-release and LC-MSn analysis of glycopeptides

Journal article
Authors Jonas Nilsson
Göran Larson
Published in Methods in Molecular Biology
Volume 951
Pages 79-100
ISSN 1064-3745
Publication year 2013
Published at Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
Pages 79-100
Language en
Links dx.doi.org/10.1007/978-1-62703-146-...
Keywords CID, ECD, Glycopeptide, Periodate oxidation, Sialic acid, article, cerebrospinal fluid, collisionally activated dissociation, deglycosylation, electron capture detection, electrospray mass spectrometry, human, peptide analysis, priority journal, protein database, reversed phase liquid chromatography, tandem mass spectrometry
Subject categories Molecular biology

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., MS 3) 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 fiuid are described. © Springer Science+Business Media, LLC 2013.

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