title: Nano-HPLC-MS of glycopeptides obtained after nonspecific proteolysis.
abstract: Liquid chromatography-tandem stage mass spectrometry of glycopeptides is a powerful tool for the site-specific glycosylation analysis of glycoproteins. Using fetuin as a model substance, we describe a protocol for glycopeptide dissection using nonspecific proteolysis by proteinase K. Proteolysis is achieved using dissolved or immobilized enzyme. For glycopeptide separation three different nanoHPLC separation principles are compared, namely hydrophilic interaction liquid chromatography (HILIC), C18-reverse phase (RP), and graphitized carbon HPLC. Chromatographically resolved glycopeptides are analyzed by nano-electrospray ionization multistage mass spectrometry for identification of the glycan as well as the peptide moiety. Using this approach, site-specific information on protein glycosylation is obtained.
authors.affiliation: 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: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D030562:Databases, Protein; D019286:Endopeptidase K / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D036103:Nanotechnology / Q000379:methods*; D059748:Proteolysis; D012996:Solutions; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004800:Enzymes, Immobilized; D006020:Glycopeptides; D012996:Solutions; D019286:Endopeptidase K