Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 39, Pages 14011-14016Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0406095101
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- NIGMS NIH HHS [R37 GM016609, GM16609, R01 GM016609] Funding Source: Medline
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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 (approximate to6 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 25degreesC to 125degreesC, 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 a-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.
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