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

• 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

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

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)