title: Chemical in-gel deglycosylation of O-glycoproteins improves their staining and mass spectrometric identification.
abstract: Heavily O-glycosylated membrane-tethered or secreted proteins often escape identification by gel-based proteomics due to weak staining and low identification rates in MS/MS. The present protocol refers to a chemical in-gel de-O-glycosylation of proteins based on repeated oxidation/elimination of glycans leaving the protein backbone intact at the gel position of the native glycoprotein. On restaining prior to spot picking, the deglycosylated proteins are detectable at increased staining intensities when applying fluorescent dyes or silver stains. Evidence shows that de-O-glycosylation of proteins in gels is efficient and does not introduce structural artifacts into the protein backbones. In-gel trypsin digestion of deglycosylated proteins, such as human glycophorin A, revealed strongly enhanced sequence coverage in LC-ESI MS/MS. The protocol is applicable in 1D and 2D gel settings within one working day.
authors.affiliation: Medical Faculty, Institute of Biochemistry II, University of Cologne, Köln, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Bellwied
authors.forename: Simon
authors.initials: S
authors.lastname: Staubach
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
mesh_terms: D000595:Amino Acid Sequence; D004396:Coloring Agents / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
chemical_list: D004396:Coloring Agents; D006020:Glycopeptides; D006023:Glycoproteins; D010446:Peptide Fragments; D014357:Trypsin
keywords: Gel-based proteomics; Glycoproteomics; O-glycoprotein