title: Protein glycosylation analysis by HILIC-LC-MS of Proteinase K-generated N- and O-glycopeptides.
abstract: 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.
authors.affiliation: Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Gerhild
authors.initials: G
authors.lastname: Zauner
authors.forename: Carolien A M
authors.initials: CA
authors.lastname: Koeleman
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D019286:Endopeptidase K / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D036103:Nanotechnology; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D006020:Glycopeptides; D011506:Proteins; D019286:Endopeptidase K