Evaluating the performance of photon- and electron-based fragmentation methods in Omnitrap-LCMS analysis of N-glycopeptides

To date, collision-induced dissociation and methods based on electron transfer dissociation are considered the standard 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 (such as electron ionisation dissociation, EID) have been shown to be useful for the analysis of glycopeptides, producing rich information on the glycopeptide structure, including types of glycosidic linkages. We evaluated ultraviolet photodissociation (UVPD), electron ionization dissociation (EID), electron capture dissociation (ECD), and activated-ion ECD (AI-ECD) using an Orbitrap-Omnitrap hybrid for LC-MS analysis of complex N-glycopeptides. Both UVPD and EID generated extensive peptide, glycosidic, and cross-ring fragments, enabling detailed structural characterization. While ECD alone produced few glycopeptide identifications, AI-ECD significantly improved yields through supplemental vibrational activation. UVPD and EID achieved comparable identification efficiencies to stepped collisional dissociation and provided additional linkage information. These results establish the Omnitrap as a powerful platform for comprehensive glycoproteomic analysis and highlight the need for enhanced computational tools to interpret complex UVPD and EID spectra.