New Energy Setup Strategy for Intact N‑Glycopeptides Characterization Using Higher-Energy Collisional Dissociation

With the optional setting of multiple stepped collisional energies (NCEs), higher-energy collisional dissociation (HCD) as available on Orbitrap instruments is a widely adopted dissociation method for intact N-glycopeptides characterization, where peptide backbones and N-glycan moieties are selectively fragmented at high and low NCEs, respectively. Initially, a dependent setting of a central value plus minus a variation is available to the users to set up NCEs, and the combination of 30 ± 10% to give the energies 20%/30%/40% has been mostly adopted in the literature. With the recent availability of an independent NCEs setup, we found that the combination of 20%/30%/30% is better than 20%/30%/40%; in the analysis of complex intact N-glycopeptides enriched from gastric cancer tissues, total IDs with spectrum-level FDR ≤ 1%, site-specific IDs with site-determining fragment ions, and structure-specific IDs with structure-diagnostic fragment ions were increased by 42% (4,767 → 6,746), 57% (599 → 942), and 97% (1771 → 3495), respectively. This finding will benefit all the coming N-glycoproteomics studies using HCD as the dissociation method.