title: Site-Specific N- and O-Glycopeptide Analysis Using an Integrated C18-PGC-LC-ESI-QTOF-MS/MS Approach.
abstract: The vast heterogeneity of protein glycosylation, even of a single glycoprotein with only one glycosylation site, can give rise to a set of macromolecules with different physicochemical properties. Thus, the use of orthogonal approaches for comprehensive characterization of glycoproteins is a key requirement. This chapter describes a universal workflow for site-specific N- and O-glycopeptide analysis. In a first step glycoproteins are treated with Pronase to generate glycopeptides containing small peptide sequences for enhanced glycosylation site assignment and characterization. These glycopeptides are then separated and detected using an integrated C18-porous graphitized carbon-liquid chromatography (PGC-LC) setup online coupled to a high-resolution electrospray ionization (ESI)-quadrupole time-of-flight (QTOF)-mass spectrometer operated in a combined higher- and lower-energy CID (stepping-energy CID) mode. The LC-setup allows retention of more hydrophobic glycopeptides on C18 followed by subsequent capturing of C18-unbound (glyco)peptides by a downstream placed PGC stationary phase. Glycopeptides eluted from both columns are then analyzed within a single analysis in a combined data acquisition mode. Stepping-energy CID results in B- and Y-ion fragments originating from the glycan moiety as well as b- and y-ions derived from the peptide part. This allows simultaneous site-specific identification of the glycan and peptide sequence of a glycoprotein.
authors.affiliation: Division of BioAnalytical Chemistry, VU University Amsterdam, De Boelelaan 1083, Amsterdam, 1081, HV, The Netherlands.|Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, Leiden, 2333, ZA, The Netherlands.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Stavenhagen
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, 14424, Germany.|Department of Biology, Chemistry, Pharmacy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, 14195, Germany.
authors.forename: Hannes
authors.initials: H
authors.lastname: Hinneburg
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids andInterfaces, Am Mühlenberg 1, Potsdam, 14476, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kolarich
authors.affiliation: Division of BioAnalytical Chemistry, VU University Amsterdam, De Boelelaan 1083, Amsterdam, 1081, HV, The Netherlands. m.wuhrer@lumc.nl.|Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, Leiden, 2333, ZA, The Netherlands. m.wuhrer@lumc.nl.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D000478:Alkylation; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D054794:Glycomics / Q000379:methods; D006020:Glycopeptides / Q000032:analysis*; D006023:Glycoproteins / Q000737:chemistry*; D006108:Graphite / Q000737:chemistry*; D006801:Humans; D011134:Polysaccharides / Q000032:analysis*; D016062:Porosity; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D006108:Graphite
keywords: C18-PGC-LC; Glycoproteomics; Mass spectrometry; N-glycopeptide; O-glycopeptide; Porous graphitized carbon; QTOF-MS; Stepping-energy CID