Characterization of glycopeptides using a stepped higher-energy C-trap dissociation approach on a hybrid quadrupole...

Accurate characterization of glycopeptides without a prior glycan cleavage could provide valuable information on site-specific glycosylation, which is critical to reveal the biological functions of protein glycosylation. However, due to the distinct nature of oligosaccharides and ploypeptides, it is usually difficult to effectively fragment glycopeptides in mass spectrometry analysis.

Here we applied a stepped normalized collisional energy (NCE) approach, which is able to combine fragment ions from three different collision energies, in a hybrid quadrupole orbitrap (Q Exactive Plus) to characterize glycopeptides. A systematic evaluation was firstly performed to find optimal NCE values for the fragmentation of glycan chains and peptide backbones from glycopeptides. Guided by the results of the systematic evaluation, the stepped NCE method was optimized and employed to analyze glycopeptides enriched from human serum.

The stepped NCE approach was found to effectively fragment both the glycan chains and peptide backbones from glycopeptides and record these fragments in a single MS/MS spectrum. In comparison with the regular HCD methods, the stepped NCE method identified more glycopeptides with higher scores from human serum samples.

Our studies demonstrate the capability of stepped NCE for the effective characterization of glycopeptides on a large scale.