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 Akkermansia 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 the
OpeRATOR digestion of four known O-glycoproteins. Our data show that
over 50% of O-glycopeptides in our sample 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.