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

• Published in: Journal of separation science Vol. 33; no. 6-7; pp. 903 - 910
• Main Authors: Zauner, Gerhild, Koeleman, Carolien A.M, Deelder, André M, Wuhrer, Manfred
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 01.03.2010; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Backbone; Chromatography, Liquid - methods; Elution; Endopeptidase K - chemistry; Glycopeptide; Glycopeptides; Glycopeptides - chemistry; Glycosylation; Hydrophilic interaction liquid chromatography; Mathematical models; NanoESI-MS; Nanostructure; Nanotechnology; Peptides; Proteinase; Proteinase K; Proteins; Proteins - chemistry; Spectrometry, Mass, Electrospray Ionization - methods
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.200900850

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.