title: Site-Specific Protein N- and O-Glycosylation Analysis by a C18-Porous Graphitized Carbon-Liquid Chromatography-Electrospray Ionization Mass Spectrometry Approach Using Pronase Treated Glycopeptides.
abstract: The analysis of N- and O-glycopeptides remains challenging due to the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of the glycoprotein. Trypsin is by far the most commonly used protease in glycoproteomic studies; however, it often results in long peptides that can harbor more than one glycan which may hamper site identification. The use of unspecific proteases such as Pronase can largely overcome this problem by generating glycopeptides with a small peptide portion. While the resulting glycopeptides are very useful for tandem mass spectrometric investigation, the analysis with conventional 1D-LC-ESI-MS/MS approaches can lead to incomplete glycosylation coverage because of the very heterogeneous physicochemical properties of the glycopeptides depending on the peptide sequence as well as the size and charges of the glycan moiety. Here, we describe a universal workflow for site-specific N- and O-glycopeptide analysis of Pronase treated glycoproteins with integrated, sequential C18 reverse phase and porous graphitized carbon-LC-ESI-QTOF-MS/MS employing a combination of lower- and enhanced-energy collision-induced dissociation. The approach was evaluated on glycoprotein standards and also applied to investigate the glycosylation of human IgG3 providing details on the hitherto uncharacterized glycosylation site Asn392 of the CH3 domain. This analytical tool can be applied to a variety of glycoproteins for site-specific N- and O-glycopeptide analysis, resulting in a good glycopeptide coverage within a single sample run and, thus, requiring only small amounts of sample.
authors.affiliation: Division of BioAnalytical Chemistry, VU University Amsterdam , De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Stavenhagen
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center , P.O. Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Rosina
authors.initials: R
authors.lastname: Plomp
authors.affiliation: Division of BioAnalytical Chemistry, VU University Amsterdam , De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.|Center for Proteomics and Metabolomics, Leiden University Medical Center , P.O. Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D002244:Carbon / Q000737:chemistry*; D002853:Chromatography, Liquid; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D010316:Particle Size; D016062:Porosity; D011402:Pronase / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013499:Surface Properties
chemical_list: D006020:Glycopeptides; D002244:Carbon; D011402:Pronase