HCD fragmentation mostly breaks glycosidic bonds, whereas ETD preserves the glycan attachment and fragments the peptide backbone, providing more complete peptide sequence information
Current MS/MS acquisition strategies for glycopeptide analysis rely on the acquisition of MS/MS spectra for all precursor ions
In this study, HCD was employed to generate glycan oxonium ions and trigger an ETD spectrum in a data-dependent manner
HCD presents the sugar signatures within the low m/z range, which are otherwise lost as a result of the one-third rule of ion trap fragmentation (44)
Glycopeptides with terminal HexNAc generate typically an m/z 204.0864 oxonium ion and its fragments at m/z 168.0653 and 138.0550
The oxonium ion and its fragments are measured with the high mass accuracy of the Orbitrap analyzer, and the unambiguous identification of the glycan oxonium ion generated by the HCD scan serves as a diagnostic marker for glycopeptides
This approach was compared against conventional HCD-alt-ETD scans using a complex biological sample
The HCD-alt-ETD preferentially detects higher charged and higher intensity precursor ions than HCD-pd-ETD
This might be because (i) a higher charge increases ETD fragmentation efficiency, resulting in more identified glycopeptides ; (ii) high-charged precursors did not produce HCD spectra of sufficient quality to trigger ETD based on the diagnostic oxonium ions; or (iii) more abundant peptides were selected in HCD-alt-ETD because the instrument duty cycle is less efficient than in HCD-pd-ETD
Overall, the combination of multiple MS methods used in our study provides greater confidence in the identification of glycopeptides than studies relying on a single approach and offers complementary advantages in the assessment of the glycoproteome, notably, the simultaneous identification of the peptide sequence , the glycosylation site , and the glycan com position