Fundamentals of Electron Capture Dissociation
The free radical cascade.
In an experiment designed to attempt to sequence cyclic peptides using Electron Capture Dissociation (ECD), a large number of unexpected peaks showed up which could only arise from multiple cleavages along the peptide backbone. This observation apparently violated the energy rules since ECD only generates ~4-6 eV, which is insufficient to cleave more than one bond. After very difficult data analysis, it appeared that the fragments were being generated by multiple free radical cleavages – which led to the proposal of a new fragmentation mechanism, the free radical cascade.
These results are discussed here:
Leymarie, N.; Costello, C. E.; O’Connor, P. B. Electron capture dissociation initiates a free radical reaction cascade J. Am. Chem. Soc. 2003, 125, 8949-8958 Link.
It seemed likely that this free radical cascade mechanism would also apply to linear peptides. So a series of experiments was proposed which would test that hypothesis.
1. Deuterium labeling of alpha carbon positions in peptides should result in deuterium scrambling if there is a long-lived radical intermediate.
Result: deuterium scrambling was detected, even though the kinetic isotope effect would substantially reduce it. Furthermore, D-scrambling percentages under ECD conditions were 10-fold higher than under SORI-CAD conditions.
2. Radical intermediates might lave lifetimes sufficient to allow a double-resonance experiment to eject them prior to final dissociation and formation of the observed fragments, thus putting limits on the lifetimes of these intermediates.
Result: Double resonance showed that many of these long lived radical intermediates have millisecond lifetimes or more. This was shown on substance p, fibrinopeptide, some synthetic peptides, and ubiquitin. Ubiquitin 11+ showed very little depletion of the fragment ions, while Ubiquitin 7+ showed extesnive depletion indicating that the former is unfolded while the latter highly folded.
3. Addition of a radical trap moiety to a peptide should substantially change the fragmentation pattern by locking the radical in place and preventing the cascade.
Result: Use of a coumarin tag as a radical trap eliminated backbone fragmentation and changed the sidechain fragmentation substantially. Some evidence of formation of a pi-stack in the gas phase was observed.
Each of these experiments has been done and are currently in various stages of publication. #1 is in press with the Journal of the American Society for Mass Spectrometry.
