Abstract We report O-Pair Search, a new approach to identify O-glycopeptides and localize O-glycosites. Using paired collision- and electron-based dissociation spectra, O-Pair Search identifies O-glycopeptides using an ion-indexed open modification search and localizes O-glycosites using graph theory and probability-based localization. O-Pair Search reduces search times more than 2,000-fold compared to current O-glycopeptide processing software, while defining O-glycosite localization confidence levels and generating more O-glycopeptide identifications. O-Pair Search is freely available: https://github.com/smith-chem-wisc/MetaMorpheus .

O-Pair Search with MetaMorpheus for O-glycopeptide Characterization