Quantification and Site-Specific Analysis of Co-occupied N- and O-Glycopeptides

Abstract

Protein glycosylation is a complex post-translational modification that is generally classified as N-or O-linked. Sitespecific analysis of glycopeptides is accomplished with a variety of fragmentation methods, depending on the type of glycosylation being investigated and the instrumentation available. For instance, collisional dissociation methods are frequently used for Nglycoproteomic analysis with the assumption that one N-sequon exists per tryptic peptide. Alternatively, electron-based methods are preferable for O-glycosite localization. However, the presence of simultaneously N-and O-glycosylated peptides could suggest the necessity of electron-based fragmentation methods for Nglycoproteomics, which is not commonly performed. Thus, we quantified the prevalence of N-and O-glycopeptides in mucins and other glycoproteins. A much higher frequency of co-occupancy within mucins was detected whereas only a negligible occurrence occurred within nonmucin glycoproteins. This was demonstrated from analyses of recombinant and/or purified proteins, as well as more complex samples. Where co-occupancy occurred, O-glycosites were frequently localized to the Ser/Thr within the N-sequon. Additionally, we found that O-glycans in close proximity to the occupied Asn were predominantly unelaborated core 1 structures, while those further away were more extended. Overall, we demonstrate electron-based methods are required for robust site-specific analysis of mucins, wherein co-occupancy is more prevalent. Conversely, collisional methods are generally sufficient for analyses of other types of glycoproteins.