Simply Extending the Mass Range in Electron TransferHigher Energy Collisional Dissociation Increases Confidence in N-GlycopeptideIdentification

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 MS/MS m/z range from 2000 to 4000 during 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 glyopeptides 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 datasets containing all classes of glycopeptides; high mannose, hybrid, and complex, measured with stand...