To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the ␣-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the ␣-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c·, and they also show c·-1 peaks in the deuterated peptides only. The presence of the c·-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H· and ϪCO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments. (J Am Soc Mass Spectrom 2006, 17, 576 -585)

To further test the hypothesis that electron capture dissociation (ECD) involves long-lived radical intermediates and radical migration occurs within these intermediates before fragmentation, radical trap moieties were attached to peptides with the assumption that they would reduce fragmentation by decreasing the mobility of the radical. Coumarin labels were chosen for the radical traps, and unlabeled, singly-labeled, and doubly-labeled Substance P were analyzed by ECD. The results demonstrated a correlation between the number and position of tags on the peptide and the intensity of side-chain cleavages observed, as well as an inverse correlation between the number of tags on the peptide and the intensity of backbone cleavages. Addition of radical traps to the peptide inhibits backbone cleavages, suggesting that either radical mobility is required for these cleavages, or new noncovalent interactions prevent separation of backbone cleavage fragments. The enhancement of side-chain cleavages and the observation of new side-chain cleavages associated with aromatic groups suggest that the gas-phase conformation of this peptide is substantially distorted from untagged Substance P and involves previously unobserved interactions between the coumarin tags and the phenylalanine residues. Furthermore, the use of a double resonance (DR)-ECD experiment showed that these side-chain losses are all products of long-lived radical intermediate species, which suggests that steric hindrance prevents the coumarin-localized radical from interacting with the backbone while simultaneously increasing the radical rearrangements with the side E lectron capture dissociation (ECD), a relatively new tandem mass spectrometry fragmentation technique, plays a useful role in protein sequencing and identification/localization of posttranslational modifications due to the backbonedirected nature of the cleavages generated. ECD targets NOC ␣ bonds [1, 2] and disulfide bonds, [3], but preserves post-translational modifications [4] such as phosphorylation [5, 6] and glycosylation [7,8]. ECD is capable of discriminating between conformers and, thus, allows for direct observation and analysis of intermediate and unfolded states [9 -11]. ECD also provides complementary data to collision activated dissociation (CAD) [3].ECD involves reactions between multiply charged peptide ions and low-energy electrons [1,3], which result in rapid backbone fragmentation and in the creation of radicals that further propagate intramolecularly and induce numerous cleavages throughout the peptide [12]. Generally, ECD results in three types of cleavages: backbone, side-chain groups, and small molecule losses. Although backbone cleavages N-terminus to proline have never been reported [13], one of the most important characteristics of ECD is its relative non-specificity [3]. In spite of its clear utility, the mechanism of ECD is still under vigorous debate [2, 9, 12, 14 -18].Currently, it is believed that the primary mechanism of ECD involves nonergodic backbone cleavage at N...