Electron-based dissociation is needed for O-glycopeptides derived from OpeRATOR proteolysis

The recently described O-glycoprotease OpeRATOR presents exciting opportunities for O-glycoproteomics. This bacterial enzyme purified from A. muciniphila cleaves N-terminally to serine and threonine residues that are modified with (preferably asialylated) O-glycans, providing orthogonal cleavage relative to canonical proteases (e.g., trypsin) to improve O-glycopeptide characterization with tandem mass spectrometry (MS/MS). O-glycopeptides with a modified N-terminal residue, such as those generated by OpeRATOR, present several potential benefits, perhaps the most notable being de facto O-glycosite localization without the need of glycan-retaining fragments in MS/MS spectra. Indeed, O-glycopeptides modified exclusively at the N-terminus would enable O-glycoproteomic methods to rely solely on collision-based fragmentation rather than electron-driven dissociation, but modified peptides would need to reliably contain only a single O-glycosite. Here we characterize the number of O-glycosites (i.e., missed cleavages) that are present in OpeRATOR-derived O-glycopeptides using methods that combine collision- and electron-based fragmentation. Our data show that over 50% of O-glycopeptides generated from combined digestion using OpeRATOR and trypsin contain multiple O-glycosites, indicating that collision-based fragmentation is not sufficient for OpeRATOR-centric studies and that electron-driven dissociation remains a requirement for site-specific O-glycopeptide characterization.