Method for investigation of oligosaccharides from glycopeptides: direct determination of glycosylation sites in proteins.
Characterization of glycopeptides has become an important tool toward a better understanding of the molecular details in carbohydrate-protein interactions. In this approach, oligosaccharides are commonly not detectable under mass spectrometric conditions because of ionization suppression by deglycosylated peptides. Their composition is only deduced from the mass differences between glycopeptides and corresponding deglycosylated peptides. Here, we describe how carbohydrates can be easily detected in the PNGase-treated samples and structurally investigated next to the peptides. The efficacy of this method is demonstrated through the analysis of tryptic glycopeptides obtained from human IgG. Following deglycosylation with PNGaseF and derivatization with phenylhydrazine, MALDI spectra produced ion peaks of labeled oligosaccharides and deglycosylated peptides. The relative abundances of individual oligosaccharides were consistent with those of the glycopeptides. MALDI-MS/MS provided useful data for the structural elucidation of oligosaccharides, including the assignment of dominant isomers and glycosylation sites in peptides. MALDI-MS/MS fragmentation patterns of deglycosylated peptide ions indicated glycosylation sites at asparagine 297 and 299. The observed peptide of the composition ADQTVYR, described for the first time in this study, indicated new glycosylation sites in IgG1 human myeloma plasma.
Lattová
Erika
E
Chemistry Department and Physics and Astronomy Department, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. lattovae@cc.umanitoba.ca
Kapková
Petra
P
Krokhin
Oleg
O
Perreault
Hélène
H
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D009844:Oligosaccharides / Q000032:analysis*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D006020:Glycopeptides; D009844:Oligosaccharides