Detection of individual glycosylation sites on glycoproteins

In this unit, glycopeptides generated by endopeptidase digestion are first separated by reversed-phase chromatography. The presence of hydrophilic, negatively charged oligosaccharides shortens retention times, causing glycopeptides to elute in considerably broader peaks than do peptides, so by following the elution profile either radiochemically or colorimetrically, the peaks corresponding to unique glycopeptides can be identified. With proper controls, the number of peaks will correspond to the number of different glycosylation sites. The eluted fractions are suitable for analysis by lectin chromatography, and the peptide sugar linkage can be defined either by endoglycosidase digestion or chemical cleavage. Oligosaccharides freed from the peptide according to the methods described in this unit can be characterized by size or charge, techniques not generally applicable with glycopeptides.