Protein glycosylation analysis by HILIC-LC-MS of Proteinase K-generated N- and O-glycopeptides

Zauner, G.; Koeleman, C.A.M.; Deelder, A.M.; Wuhrer, M. (2010)

Article / Letter to editor

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; MS3 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.

• Glycopeptide Glycosylation Hydrophilic interaction liquid chromatography NanoESI-MS Proteinase K hydrophilic-interaction chromatography mass-spectrometry linked glycosylation zwitterionic type digestion pronase identification glycoproteins separation sites

• All authors: Zauner, G.; Koeleman, C.A.M.; Deelder, A.M.; Wuhrer, M.
• Date: 2010-04-30
• Journal: Journal of Separation Science
• Volume: 33
• Issue: 6-7
• Pages: 903 - 910
• DOI: doi:10.1002/jssc.200900850