Oligosaccharides play important roles in diverse biological processes, and alterations in them are thought to be associated with some diseases. The elucidation of glycosylation-related diseases will require glycomic approaches based on qualitative and quantitative analyses of glycosylation. We have studied the various analytical methods to determine carbohydrate heterogeneity, glycosylation sites, and site-specific glycosylation in glycoproteins by using LC/MS.
LC/MS/MS of glycopeptides
Mass spectrometric peptide/glycopeptide mapping is frequently used for the determination of glycosylation sites and site-specific glycosylation. In-source fragmentation and precursor ion scanning, which monitor sugar-specific oxonium ions, are employed to extract glycopeptide ions from a large number of peptide ions, however, additional analyses are required in order to obtain peptide fragmentation patterns or precursor ions. Using automated data-dependent MS/MS as an alternative method to distinguish glycopeptide ions from peptide ions and to obtain both the peptide's and sugar's fragment ions, we have successfully determined the site-specific glycosylation of some glycoproteins.
LC/MS of oligosaccharides
Glycoproteins exist in heterogeneous forms because of the various combinations of oligosaccharides including isomers. We have developed mass spectrometric oligosaccharide profiling using a graphitized carbon column, which can separate oligosaccharides based on subtle differences in branch, position, and linkage isomers with volatile solution. LC/MS provides structural information based on chromatographic behavior and molecular mass. This method is expected to be a powerful tool for glycome analysis. In this study we apply oligosaccharide profiling to differential analyses of N-linked oligosaccharides between treated and untreated cells.
