title: Characterization of Protein N-Glycosylation by Analysis of ZIC-HILIC-Enriched Intact Proteolytic Glycopeptides.
abstract: Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction (SPE) combined with direct-infusion nanoESI mass spectrometry (MS) and tandem MS/MS is a well-suited method for the analysis of protein N-glycosylation. A site-specific characterization of N-glycopeptides is achieved by the combination of proteolytic digestions employing unspecific proteases, glycopeptide enrichment by use of ZIC-HILIC SPE, and subsequent mass spectrometric analysis. The use of thermolysin or a mixture of trypsin and chymotrypsin leads per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. As a result of their higher hydrophilicity N-glycopeptides comprising short peptide backbones are preferably accumulated by the ZIC-HILIC-based separation procedure. By employing this approach complications associated with low ionization efficiencies of N-glycopeptides resulting from signal suppression in the presence of highly abundant nonglycosylated peptides can be largely reduced. Here, we describe a simple protocol aimed at the enrichment of N-glycopeptides derived from in-solution and in-gel digestions of SDS-PAGE-separated glycoproteins preceding mass spectrometric analysis.
authors.affiliation: Institute for Hygiene, University of Münster, Robert-Koch-Strasse 41, 48149, Münster, Germany.
authors.forename: Gottfried
authors.initials: G
authors.lastname: Pohlentz
authors.affiliation: Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359, Bremen, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Marx
authors.affiliation: Institute for Hygiene, University of Münster, Robert-Koch-Strasse 41, 48149, Münster, Germany. mmormann@uni-muenster.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Mormann
mesh_terms: D002845:Chromatography / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods*; D059748:Proteolysis
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins
keywords: CID; Glycopeptides; In-gel digestion; In-solution digestion; N-glycosylation; NanoESI MS