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Protein glycosylation analysis by HILIC‐LC‐MS of Proteinase K‐generated N ‐ and O ‐glycopeptides
Author(s) -
Zauner Gerhild,
Koeleman Carolien A. M.,
Deelder André M.,
Wuhrer Manfred
Publication year - 2010
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200900850
Subject(s) - glycopeptide , chemistry , fetuin , glycosylation , glycan , peptide , glycoprotein , hydrophilic interaction chromatography , chromatography , oligosaccharide , biochemistry , mass spectrometry , high performance liquid chromatography , antibiotics
Analysis of protein glycosylation is essential in order to correlate certain disease types with oligosaccharide structures on proteins. Here, a method for the MS characterization of site‐specific protein glycosylation is presented. Using asialofetuin and fetuin as model substances, a protocol for glycopeptide dissection was developed based on unspecific proteolysis by Proteinase K. The resulting glycopeptides were then resolved by nanoscale hydrophilic interaction liquid chromatography‐electrospray multistage MS. The early elution range of O ‐glycopeptides was clearly separated from the late elution range of N ‐glycopeptides. Glycopeptides were analyzed by ion trap‐MS/MS, which revealed fragmentations of glycosidic linkages and some peptide backbone cleavages; MS 3 spectra predominantly exhibited cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N ‐ and O ‐glycan attachment sites of fetuin could be confirmed; moreover using our method, the occupation of a new, additional O ‐glycosylation site serine 296 was found. In conclusion, this approach appears to be a valuable technique for in‐depth analysis of the site‐specific N ‐glycosylation and O ‐glycosylation of individual glycoproteins.