Abstract
Great advances have been made in mass spectrometric data interpretation for intact glycopeptide analysis. However, accurate identification of intact glycopeptides and modified saccharide units at the site-specific level and with fast speed remains challenging. Here, we present a glycan-first glycopeptide search engine, pGlyco3, to comprehensively analyze intact N- and O-glycopeptides, including glycopeptides with modified saccharide units. A glycan ion-indexing algorithm developed for glycan-first search makes pGlyco3 5–40 times faster than other glycoproteomic search engines without decreasing accuracy or sensitivity. By combining electron-based dissociation spectra, pGlyco3 integrates a dynamic programming-based algorithm termed pGlycoSite for site-specific glycan localization. Our evaluation shows that the site-specific glycan localization probabilities estimated by pGlycoSite are suitable to localize site-specific glycans. With pGlyco3, we confidently identified N-glycopeptides and O-mannose glycopeptides that were extensively modified by ammonia adducts in yeast samples. The freely available pGlyco3 is an accurate and flexible tool that can be used to identify glycopeptides and modified saccharide units.
Highlights
Great advances have been made in mass spectrometric data interpretation for intact glycopeptide analysis
PGlyco 2.0 supports only the search against the normal mammalian N-glycans present in GlycomeDB33 with stepped collision energy HCD (sceHCD) spectra; it is difficult for users to apply it to analyze customized glycans or modified saccharide units
As the Y-complementary ion mass does not contain the peptide mass, it enables us to search the glycans before peptides are identified
Summary
Great advances have been made in mass spectrometric data interpretation for intact glycopeptide analysis. Modern glycopeptide search engines should consider site-specific glycan localization (SSGL), as ETxxD techniques have been used widely in glycoproteomics Some works, such as the ‘Delta Mod score’ of Byonic and ‘SLIP’34 of Protein Prospector, extended the site localization algorithms from traditional PTMs to glycosylation, and assessed the localization reliabilities. PGlyco applies the glycan-first search strategy to accurately identify glycopeptides It uses canonicalization-based glycan databases to support the modified saccharide unit analysis and implements a glycan ion-indexing technique to accelerate the search for glycans. We validated the aH search results on yeast with N-glycome data and 15N-/13C-labeled glycopeptide data This analysis further demonstrates the reliability and flexibility of pGlyco for intact glycopeptide and modified saccharide unit identification
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