Evaluating the Performance of Photon- and Electron-Based Fragmentation Methods in Omnitrap-LCMS Analysis of <i>N</i> -Glycopeptides

High Resolution Image Download MS PowerPoint Slide To date, collision-induced dissociation and methods based on electron transfer dissociation are considered the ‘best’ approaches for the mass spectrometry analysis of N - and O -glycopeptides, respectively, allowing for identification of both peptide and glycan compositions. In recent years, alternative fragmentation methods such as ultraviolet photodissociation (UVPD) and more energetic versions of electron-based techniques (e.g., electron ionization dissociation, EID) have been shown to be useful for analysis of glycopeptides, producing rich information on the glycopeptide structure, including types of glycosidic linkages. We evaluated UVPD, EID, electron capture dissociation (ECD), and activated-ion ECD (AI-ECD) using an Orbitrap–Omnitrap hybrid for LCMS analysis of complex N -glycopeptides. Both UVPD and EID generated extensive peptide, glycosidic, and cross-ring fragments, complementary to standard collisional dissociation, and achieved comparable identification efficiencies. While ECD alone produced few glycopeptide identifications, AI-ECD significantly improved yields through supplemental vibrational activation. These results establish the Omnitrap, and its ability to house multiple fragmentation approaches, as a powerful platform for comprehensive glycoproteomic analysis and highlight the continuing need for enhanced computational tools to interpret complex UVPD and EID spectra.