RESULTS
Traditional HDX-MS analysis of a glycoprotein’s conformational dynamics often leaves large information gaps because of challenges in analyzing the deuterium uptake of glycopeptides. During our traditional HDX-MS analysis of SARS-CoV-2 spike protein, we found significant gaps in sequence coverage and a lack of information in many areas of interest. To fill these sequence coverage gaps, we incorporated signature ion-triggered EThcD into HDX-MS analysis (final spike (glyco)peptide coverage in Figure S2).
Identification of glycopeptides used Fourier-transform mass spectrometry (FTMS) scans to detect precursor ions, and those above an intensity threshold were selected for high-energy collision-induced dissociation (HCD) FTMS/MS scans. This relatively energetic fragmentation yields abundant b- and y-ions for spectral matching to peptide sequences, but can also produce oxonium ions (such as HexNAc 204.0867 m/z , HexNAc fragment 138.0545 m/z , and Hex(2)NAc 366.1396 m/z ) diagnostic for the presence of complex glycosylation of the precursor peptide (33, 39, 43, 44). The presence of these signature oxonium ions triggered data-dependent electron transfer low-energy collision-induced dissociation (EThcD) FTMS/MS of a fresh precursor ion packet, yielding peptide fragments retaining 1 or more hexose groups or intact glycan, ideally confirming the glycosylation sequon’s location (45).