title: Protein glycosylation analyzed by normal-phase nano-liquid chromatography--mass spectrometry of glycopeptides.
abstract: A new method for the mass spectrometric characterization of site-specific protein glycosylation is presented. Glycoprotein samples were subjected to unspecific proteolysis by Pronase, resulting in glycopeptides with peptide moieties of mostly two to eight amino acids. Resulting (glyco-)peptide samples were resolved by nanoscale normal-phase liquid chromatography (LC)-online mass spectrometry (MS). Retention depended on the size of the glycan chain and allowed the separation of identical peptide moieties containing different N-glycan structures. Glycopeptides were analyzed in an ion trap instrument performing repetitive ion isolation/fragmentation cycles. While the MS/MS spectra were dominated by fragmentations of glycosidic linkages, MS(3) spectra exhibited cleavages of the peptide backbone and provided information on the peptide sequence and glycan attachment site. When applied to the model glycoproteins ribonuclease B and horseradish peroxidase (HRP), the method provided detailed insights into protein glycosylation and revealed some new features of site-specific glycosylation of HRP. Application of the method to Dolichos biflorus lectin, which has hitherto not been studied with respect to its glycosylation, identified two glycans attached alternatively to its single glycosylation site. Thus, the presented, unique combination of Pronase digestion of glycoproteins, normal-phase nano-LC, and multistage MS provides a method for the facile characterization of site-specific protein glycosylation.
authors.affiliation: Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. m.wuhrer@lumc.nl
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.forename: Carolien A M
authors.initials: CA
authors.lastname: Koeleman
authors.forename: Cornelis H
authors.initials: CH
authors.lastname: Hokke
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
mesh_terms: D001665:Binding Sites; D002853:Chromatography, Liquid / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry; D011402:Pronase / Q000378:metabolism; D012260:Ribonucleases / Q000032:analysis / Q000737:chemistry
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D010446:Peptide Fragments; D011134:Polysaccharides; D006735:Horseradish Peroxidase; D012260:Ribonucleases; C029600:ribonuclease B; D011402:Pronase