Precise, Fast and Comprehensive Analysis of Intact Glycopeptides and Monosaccharide-Modifications with pGlyco3

We presented a glycan-first glycopeptide search engine, pGlyco3, to comprehensively analyze intact N- and O-glycopeptides, including glycopeptides with monosaccharide-modifications. We developed an algorithm, termed pGlycoSite, to localize the glycosylation sites and estimate the localization probabilities. We designed a number of experiments to validate the accuracy of pGlyco3 as well as other frequently used or recently developed software tools. These experiments showed that pGlyco3 outperformed the other tools on both N- and O-glycopeptide identification accuracy especially at the glycan level, without loss of the sensitivity. pGlyco3 also achieved a superior performance in terms of search speed. As pGlyco3 was shown to be accurate and flexible for glycopeptide search with monosaccharide-modifications, we then discovered a monosaccharide-modification of Hex (or an uncommon monosaccharide) "Hex+17.027 Da" on both O-mannose and N-glycopeptides in yeast samples, and confirmed this monosaccharide based on released N-glycans and isotopic labelling data. pGlyco3 is freely available on https://github.com/pFindStudio/pGlyco3/releases.