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Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis

revised on 17.07.2020, 10:14 and posted on 17.07.2020, 10:57 by Nicholas Riley, Stacy Malaker, Carolyn Bertozzi

The recently described O-glycoprotease OpeRATOR presents exciting opportunities for O-glycoproteomics. This bacterial enzyme purified from Akkermansia (Sp). muciniphila cleaves N-terminally to serine and threonine residues that are modified with (preferably asialylated) O-glycans. This provides orthogonal cleavage relative to canonical proteases (e.g., trypsin) for improved 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 because glycan-retaining peptide fragments would not be required for localization. The caveat is that modified peptides would need to reliably contain only a single O-glycosite. Here we use methods that combine collision- and electron-based fragmentation to characterize the number of O-glycosites that are present in O-glycopeptides derived from OpeRATOR digestion of four known O-glycoproteins. 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 alone is not sufficient. Electron-based dissociation methods are necessary to capture the O-glycopeptide diversity present in OpeRATOR digestions.


R01 CA200423

K00 CA212454



Email Address of Submitting Author


Stanford University


United States

ORCID For Submitting Author


Declaration of Conflict of Interest

C.R.B. is a co-founder and Scientific Advisory Board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), and InterVenn Biosciences, and a member of the Board of Directors of Eli Lilly & Company.