Loss of Ammonia during Electron-Transfer Dissociation of Deuterated Peptides as an Inherent Gauge of Gas-Phase Hydrogen Scrambling

摘要

The application of electron-transfer dissociation (ETD) tonobtain single-residue resolution in hydrogen exchangemassnspectrometry (HX-MS) experiments has recentlynbeen demonstrated. For such measurements, it is criticalnto ensure that the level of gas-phase hydrogen scramblingnis negligible. Here we utilize the abundant loss of ammonianupon ETD of peptide ions as a universal reporternof positional randomization of the exchangeable hydrogensn(hydrogen scrambling) during HX-ETD experiments. Wenshow that the loss of ammonia from peptide ions proceedsnwithout depletion of deuterium when employing optimizednmild electrospray ion source settings for the HX-ETDnanalysis of a selectively labeled model peptide and peptidesnderived from fully labeled u00012-microglobulin. Hydrogennscrambling, as induced by excessive vibrationalnexcitation of peptide ions during harsh declusteringnconditions, is easily detected by a depletion of deuteriumnwhen deuterated ammonia is lost from peptidesnduring ETD. This straightforward method requires nonmodifications to the experimental workflow and has thengreat advantage that the occurrence of hydrogen scramblingncan be directly detected in the actual peptidesnanalyzed in the HX-ETD experiment.