Nonergodic and conformational control of the electron capture dissociation of protein cations.

Abstract

Electron capture dissociation (ECD) MS is proving to be unusually valuable for "top down" protein sequencing and identification/localization of posttranslational modifications, because the ECD product ions can represent cleavages between most of a protein's amino acids. As proposed, this unusual reactivity results from immediate local utilization, before randomization, of much of the relatively large ( approximately 6 eV) energy from the electron reaction with the multiply charged protein ion, minimizing the effect of differences in the backbone bond dissociation energies. However, others conclude that e(-) capture produces a labile free radical species for which backbone cleavage is the lowest energy reaction. Supporting the nonergodic mechanism, ECD of ubiquitin (M + 12H)(12+) ions also yields thermalized radical (M + 12H)(11+.) ions that instead lose H. when activated. Also, the ECD spectrum of ubiquitin (M + 13H)(13+) ions is nearly unchanged by heating from 25 degrees C to 125 degrees C, demonstrating that this increase in thermal energy is small compared to the energy driving the reaction. These results support initial capture of the electron in a long-lived high-n Rydberg state, followed by internal conversion to the product valence state at an energy well above the dissociation barriers. The instantaneous conformation of the valence state is critical, with the observed products supporting an alpha-helical structure in which the protonated side chain of each basic residue is intercalated to hydrogen-bond to as many as three amide carbonyls. Activation (e.g., heat, collisions, lowered charge) can disrupt this conformation to allow additional H(+)-side-chain interactions and provide more complete sequence coverage.