Simply Extending the Mass Range in Electron Transfer Higher Energy Collisional Dissociation Increases Confidence in N-Glycopeptide Identification

Glycopeptide-centric
mass spectrometry has become a popular approach
for studying protein glycosylation. However, current approaches still
utilize fragmentation schemes and ranges originally optimized and
intended for the analysis of typically much smaller unmodified tryptic
peptides. Here, we show that by merely increasing the tandem mass
spectrometry m/z range from 2000
to 4000 during electron transfer higher energy collisional dissociation
(EThcD) fragmentation, a wealth of highly informative c and z ion
fragment ions are additionally detected, facilitating improved identification
of glycopeptides. We demonstrate the benefit of this extended mass
range on various classes of glycopeptides containing phosphorylated,
fucosylated, and/or sialylated N-glycans. We conclude that the current
software solutions for glycopeptide identification also require further
improvements to realize the full potential of extended mass range
glycoproteomics. To stimulate further developments, we provide data
sets containing all classes of glycopeptides (high mannose, hybrid,
and complex) measured with standard (2000) and extended (4000) m/z range that can be used as test cases
for future development of software solutions enhancing automated glycopeptide
analysis.