Electron transfer dissociation facilitates the measurement of deuterium incorporation into selectively labeled peptides with single residue resolution.

Mass spectrometry is routinely applied to measure the incorporation of deuterium into proteins and peptides. The exchange of labile， heteroatom-bound hydrogens is mainly used to probe the structural dynamics of proteins in solution， e.g.， by hydrogen-exchange mass spectrometry， but also to study the gas-phase structure and fragmentation mechanisms of polypeptide ions. Despite considerable effort in recent years， there is no widely established mass spectrometric method to localize the incorporated deuterium to single amino acid residues， and typically， only the overall deuterium content of peptides or proteins is obtained. The main reason for this is that CID and related techniques induce intramolecular migration of hydrogens ("hydrogen scrambling") upon vibrational excitation of the even-electron precursor ion， thus randomizing the positional distribution of the incorporated deuterium atoms before fragmentation. In contrast， decomposition of radical gas-phase peptide cations upon electron capture dissociation was recently demonstrated to proceed with a very low level of amide hydrogen scrambling. Employing model peptides developed to enable sensitive detection of hydrogen scrambling， we show in the present study that electron transfer dissociation in a 3D-quadrupole ion trap retains the site-specific solution-phase deuterium incorporation pattern and allows for localization of incorporated deuterium with single residue resolution. Furthermore， we exploit this finding to monitor how collisional activation induces proton mobility in a gaseous peptide ion at various levels of vibrational excitation.