<p>The
recently described O-glycoprotease OpeRATOR presents exciting opportunities for
O-glycoproteomics. This bacterial enzyme purified from <i>Akkermansia (Sp). muciniphila</i>
cleaves N-terminally to serine and threonine residues that are modified with (preferably
asialylated) O-glycans. This <a>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 <i>de facto</i>
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. </a>Here
we use methods that combine collision- and electron-based fragmentation to
characterize the number of <i>O-</i>glycosites
that are present in <i>O-</i>glycopeptides
derived from OpeRATOR digestion of four known <i>O-</i>glycoproteins.
Our data show that over 50% of <i>O-</i>glycopeptides
generated from combined digestion using OpeRATOR and trypsin contain multiple <i>O-</i>glycosites, indicating that collision-based
fragmentation alone is not sufficient. Electron-based dissociation methods are
necessary to capture the <i>O-</i>glycopeptide
diversity present in OpeRATOR digestions. </p>
Copper(II) Coordination Abilities of the Tau Protein's N‐Terminus Peptide Fragments: A Combined Potentiometric, Spectroscopic and Mass Spectrometric Study