biostudies-literatureSutherland EAmerican Society for Mass SpectrometryWashington Research FoundationNational Institute of General Medical SciencesNIGMS NIH HHS5606-5614https://www.ebi.ac.uk/biostudies/studies/S-EPMC12057629biostudies-literatureUnknown23(12)Tandem mass spectrometry (MS/MS) is the gold standard for intact glycopeptide identification, enabling peptide sequence elucidation and site-specific localization of glycan compositions. Beam-type collisional activation is generally sufficient for <i>N-</i>glycopeptides, while electron-driven dissociation is crucial for site localization in <i>O-</i>glycopeptides. Modern glycoproteomic methods often employ multiple dissociation techniques within a single LC-MS/MS analysis, but this approach frequently sacrifices sensitivity when analyzing multiple glycopeptide classes simultaneously. Here we explore the utility of intelligent data acquisition for glycoproteomics through real-time library searching (RTLS) to match oxonium ion patterns for on-the-fly selection of the appropriate dissociationJournal of proteome researchAutonomous Dissociation-type Selection for Glycoproteomics Using a Real-Time Library Search.PMC12057629R00 GM147304R00GM147304Russell JHBergen DMcAlister GCHuang JHuguet RZabrouskov VBarshop WDMullen CSutherland ERiley NMKothlow KCanterbury JDVeth TSfalseAutonomous Dissociation-type Selection for Glycoproteomics Using a Real-Time Library Search.Tandem mass spectrometry (MS/MS) is the gold standard for intact glycopeptide identification, enabling peptide sequence elucidation and site-specific localization of glycan compositions. Beam-type collisional activation is generally sufficient for <i>N-</i>glycopeptides, while electron-driven dissociation is crucial for site localization in <i>O-</i>glycopeptides. Modern glycoproteomic methods often employ multiple dissociation techniques within a single LC-MS/MS analysis, but this approach frequently sacrifices sensitivity when analyzing multiple glycopeptide classes simultaneously. Here we explore the utility of intelligent data acquisition for glycoproteomics through real-time library searching (RTLS) to match oxonium ion patterns for on-the-fly selection of the appropriate dissociation2024-01-01T00:00:00Z2024 Dec2026-06-10T05:17:21.071Z2026-06-10T03:07:19.018ZS-EPMC120576293953153210.1021/acs.jproteome.4c00723