Combining gel and capillary electrophoresis, nano-LC and mass spectrometry for the elucidation of post-translational modifications of Trichoderma reesei cellobiohydrolase I.
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.
Sandra
Koen
K
Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, KL Ledeganckstraat 35, B-9000 Gent, Belgium.
Stals
Ingeborg
I
Sandra
Pat
P
Claeyssens
Marc
M
Van Beeumen
Jozef
J
Devreese
Bart
B
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*
D043366:Cellulose 1,4-beta-Cellobiosidase