Journal
JOURNAL OF PROTEOME RESEARCH
Volume 3, Issue 3, Pages 556-566Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr034112b
Keywords
proteomics; post-translational modifications; mass spectrometry; HILIC; endoglycosidase; lectin affinity chromatography; glycosylation; plasma proteins
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Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopepticles, while the remaining GlcrqAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1 D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.
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