Parallel Determination of Polypeptide and Oligosaccharide Connectivities by Energy-Resolved Collison-Induced Dissociation of Protonated O-Glycopeptides Derived from Nonspecific Proteolysis

Collision-induced dissociation (CID) is by far the most broadly applied dissociation method used for tandem mass spectrometry (MS/MS). This includes MS/MS-based structural interrogation of glycopeptides for applications in glycoproteomics. The end goal of such measurements is to determine the monosaccharide connectivity of the glycan, the amino acid sequence of the peptide, and the site of glycosylation for each glycopeptide of interest. In turn, this allows inferences with respect to the glycoprofile of the intact glycoprotein. For glycopeptide analysis, CID is best known for the ability to determine glycosidic topology of the oligosaccharide group; however, CID has also been shown to produce amide bond cleavage of the polypeptide group. Whether structural information is obtained for the glycan or the peptide has been found to depend on the applied collision energy. While these energy-resolved fragmentation pathways have been the subject of several studies on N-linked glycopeptides, there remains a dearth of similar work on O-linked glycopeptides. In this study, MS/MS via CID was shown to provide substantial peptide backbone fragmentation, in addition to glycosidic fragmentation, in an energy-dependent manner. While qualitatively similar to previous findings for N-glycopeptides, the energy-resolved CID (ER-CID) of O-glycopeptides was found to be substantially more sensitive to the collision energy setting. Thus, deliberately obtaining either glycan or peptide dissociation is a more delicate undertaking for O-glycopeptides. Establishing a more complete understanding of O-glycopeptide ER-CID is likely to have a substantive impact on how O-glycoproteomic analysis is approached in the future.