O-Glycoprotein Biosynthesis: Site Localization by Edman Degradation and Site Prediction Based on Ran

The characterization of mucin-type O-glycosylation is fraught with extreme difficulty at almost every level of analysis: from difficulties in obtaining glycopeptides suitable for study, their structural heterogeneity, lack of broad acting glycosidase tools capable of simplifying the glycans, and finally the vast complexity of performing analysis on multiply glycosylated glycopeptides. This, along with a lack of known peptide sequence motif(s) for the transferases that initiate mucin-type O-glycosylation, significantly hinders our understanding of mucin-type O-glycosylation at almost every level from their biosynthesis to their biological and biophysical properties. In this chapter, the use of partial chemical deglycosylation coupled with Edman amino acid sequencing is described to quantify sites of O-glycosylation. In addition, the use of oriented random peptide substrates is described for providing the specificities of the polypeptide α-N- acetylgalactosaminyltransferases, which can be used to estimate transferase-specific sites of O-glycosylation.