Autonomous\nDissociation-type Selection for Glycoproteomics\nUsing a Real-Time Library Search

Tandem mass spectrometry (MS/MS)\nis the gold standard\nfor intact\nglycopeptide identification, enabling peptide sequence elucidation\nand site-specific localization of glycan compositions. Beam-type collisional\nactivation is generally sufficient for <i>N-</i>glycopeptides,\nwhile electron-driven dissociation is crucial for site localization\nin <i>O-</i>glycopeptides. Modern glycoproteomic methods\noften employ multiple dissociation techniques within a single LC-MS/MS\nanalysis, but this approach frequently sacrifices sensitivity when\nanalyzing multiple glycopeptide classes simultaneously. Here we explore\nthe utility of intelligent data acquisition for glycoproteomics through\nreal-time library searching (RTLS) to match oxonium ion patterns for\non-the-fly selection of the appropriate dissociation method. By matching\ndissociation method with glycopeptide class, this autonomous dissociation-type\nselection (ADS) generates equivalent numbers of <i>N-</i>glycopeptide identifications relative to traditional beam-type collisional\nactivation methods while also yielding comparable numbers of site-localized <i>O-</i>glycopeptide identifications relative to conventional\nelectron transfer dissociation-based methods. The ADS approach represents\na step forward in glycoproteomics throughput by enabling site-specific\ncharacterization of both <i>N-</i>and <i>O-</i>glycopeptides within the same LC-MS/MS acquisition.