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.