biostudies-literatureHever HNemzeti Kutat?si, Fejleszt?si ?s Innovaci?s Alap333-343https://www.ebi.ac.uk/biostudies/studies/S-EPMC10853973biostudies-literatureUnknown35(2)High confidence and reproducibility are still challenges in bottom-up mass spectrometric <i>N</i>-glycopeptide identification. The collision energy used in the MS/MS measurements and the database search engine used to identify the species are perhaps the two most decisive factors. We investigated how the structural features of <i>N</i>-glycopeptides and the choice of the search engine influence the optimal collision energy, delivering the highest identification confidence. We carried out LC-MS/MS measurements using a series of collision energies on a large set of <i>N</i>-glycopeptides with both the glycan and peptide part varied and studied the behavior of Byonic, pGlyco, and GlycoQuest scores. We found that search engines show a range of behavior between peptide-centric and glycan-centriJournal of the American Society for Mass SpectrometryCan We Boost <i>N</i>-Glycopeptide Identification Confidence? Smart Collision Energy Choice Taking into Account Structure and Search Engine.PMC10853973PD-132135FK-138678K-131762Nagy KKomka KHever HXue ARevesz AVekey KDrahos LfalseCan We Boost <i>N</i>-Glycopeptide Identification Confidence? Smart Collision Energy Choice Taking into Account Structure and Search Engine.High confidence and reproducibility are still challenges in bottom-up mass spectrometric <i>N</i>-glycopeptide identification. The collision energy used in the MS/MS measurements and the database search engine used to identify the species are perhaps the two most decisive factors. We investigated how the structural features of <i>N</i>-glycopeptides and the choice of the search engine influence the optimal collision energy, delivering the highest identification confidence. We carried out LC-MS/MS measurements using a series of collision energies on a large set of <i>N</i>-glycopeptides with both the glycan and peptide part varied and studied the behavior of Byonic, pGlyco, and GlycoQuest scores. We found that search engines show a range of behavior between peptide-centric and glycan-centri2024-01-01T00:00:00Z2024 Feb2025-04-04T13:34:05.468Z2025-04-04T13:34:05.468ZS-EPMC108539733828602710.1021/jasms.3c00375