Exploiting differential dissociation chemistries of O-linked glycopeptide ions for the localization of mucin-type protein glycosylation.

From a glycoproteomic perspective, the unambiguous localization of O-linked oligosaccharide attachment sites is fraught with analytical obstacles. Because no consensus protein sequence exists for O-glycosylation, there is potential for glycan attachment at numerous serine and threonine residues of a given protein. The well-established tendency for O-glycan attachment to occur within serine and threonine rich domains adds further complication to site-specific assignment of mucin-type glycosylation. In addition to the complexities contributed by the polypeptide chain, the O-linked carbohydrate modifications themselves are exceedingly diverse in both compositional and structural terms. This work is aimed at contributing an improved fundamental understanding of the chemistry that dictates dissociation of O-glycopeptide ions during tandem mass spectrometry (MS/MS). Infrared multiphoton dissociation (IRMPD) has been applied to an assortment of O-linked glycopeptide ions encompassing various compositions and charge states. Protonated O-glycopeptides were found to undergo a combination of glycosidic bond cleavage (complete coverage) and peptide bond cleavage (partial coverage). In contrast to previous observations of N-linked glycopeptide dissociation, the sodiated O-glycopeptides did not yield significantly different information as compared to the corresponding protonated ions. IRMPD of deprotonated O-glycosylated peptides provided informative side chain losses from nonglycosylated serine and threonine residues, which indirectly implicated sites of glycan attachment. In this manner, the combination of positive mode and negative mode MS/MS was found to provide conclusive assignment of O-glycosites.