An identification method to distinguish monomeric sugar isomers on glycopeptides

A one-step protocol for the automated flow synthesis of protected glycosylated amino acids is described using pumps with open-source controls in overall yields of 21-50%. The resulting glycosylated amino acids could be used directly in solid-phase peptide synthesis (SPPS) protocols to quickly produce glycopeptide standards. Access to a variety of stereoisomers of the sugar enabled the development of an LC-MS/MS protocol that can distinguish between peptides modified with carbohydrates having the same exact mass. This method could definitively identify fucose in an O -glycosylation site on the transmembrane protein, Notch1. A one-step automated flow protocol for the synthesis of protected glycosylated amino acids enabled the production of glycopeptide standards that were used towards the development of an LC-MS/MS protocol.