title: Combining gel and capillary electrophoresis, nano-LC and mass spectrometry for the elucidation of post-translational modifications of Trichoderma reesei cellobiohydrolase I.
abstract: N-Glycosylation of cellobiohydrolase I from the fungus Trichoderma reesei (strain Rut-C30) is studied using a combination of electrophoretic, chromatographic and mass spectrometric techniques. As four potential N-glycosylation sites and several uncharged and phosphorylated high-mannose glycans are present, a large number of glycoforms and phospho-isoforms can be expected. Isoelectric focusing both in gel and in capillary format was successfully applied for the separation of the phospho-isoforms. They were extracted in their intact form from the gel and subsequently analysed by nanospray-Q-TOF-MS, thereby making use of a powerful two-dimensional technique. Nano-LC/MS/MS on a Q-Trap MS further allowed the determination of the glycosylation sites. As a novel approach, an oxonium ion was used in precursor ion scanning for selective detection of glycopeptides containing phosphorylated high-mannose glycans.
authors.affiliation: Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, KL Ledeganckstraat 35, B-9000 Gent, Belgium.
authors.forename: Koen
authors.initials: K
authors.lastname: Sandra
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Stals
authors.forename: Pat
authors.initials: P
authors.lastname: Sandra
authors.forename: Marc
authors.initials: M
authors.lastname: Claeyssens
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
mesh_terms: D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007525:Isoelectric Focusing; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D036103:Nanotechnology; D011499:Protein Processing, Post-Translational; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D043366:Cellulose 1,4-beta-Cellobiosidase