Signature Ions Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) for Specific and Confident Glycation Site Mapping in Therapeutic Proteins.

Higher-energy collisional dissociation (HCD) is a well-established fragmentation technique in liquid chromatography tandem mass spectrometry (LC-MS/MS) and is used to study protein post translational modifications (PTMs) during peptide mapping. However, labile PTMs like glycosylation, glycation, sulfonylation, or phosphorylation tend to fragment earlier than peptide backbones under HCD. This leads to complicated MS/MS spectra, compromising data quality and downstream data interpretation. Electron-transfer/higher-energy collision dissociation (EThcD) has been used to analyze PTMs, but important components might be missed because of the increased duty cycle. To address this issue, modification-specific fragment ions formed in HCD experiments could be utilized to trigger EThcD analysis only for modified peptides. The trigger for EThcD was established by applying HCD with a high normalized collision energy, generating multiple informative Amadori derived lysine signature ions from a glycated peptide. These signature ions were then applied to trigger targeted EThcD for lysine glycation identification. This improved approach can further expand the characterization efforts of highly labile PTMs in therapeutic proteins.