Radical a-Ions in Electron Capture Dissociation: On the Origin of Species
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Radical a* ions appear in electron capture dissociation mass spectra sporadically, but sometimes with high intensity. Mechanistically, radical a ions are hypothesized to arise due to thermodynamically disadvantaged charge solvation on the backbone nitrogen (instead of carbonyl), which upon neutralization produces a hypervalent group instantly fragmenting into a radical b* and conventional y' ion. The former species is unstable and, after releasing a CO molecule, decays to an a* ion. Here we validate this scenario by direct observation of the complementarity of a* and y' ions by interrogation of an ECD MS/MS database of >10,000 doubly and >5,000 triply charged tryptic peptides. Intriguingly, the most abundant a*/y' pairs are found to come from the cleavage of the same backbone link as the most abundant c' and z* complementary ions. This result gives strong support to the “local” N-Cα bond cleavage mechanism, in which the dissociation occurs at the site of charge solvation. However, a second strong peak is observed in the c'/z* fragment distribution four residues away from the a*/y' cleavage, which supports the indirect N-Cα bond cleavage mechanism. The size distribution of a ions from doubly (but not triply!) charged precursors shows deficit of a3 ions, and possibly a6 ions.
Key wordsPeptide fragmentation Bond cleavage mechanism
Unlike the N–Cα bond cleavage mechanism that is still under intensive debates, the scenario (1) of the \( a \cdot \) ion formation process via the sequential C–N/Cα–C bond cleavage has never been challenged in peer-reviewed literature, and is widely accepted. This, however, does not remove the need to validate (1) by direct observations. One such observation could be the complementarity of the \( a \cdot \) and y' fragments produced in a single bond cleavage event, like the c' and \( z \cdot \) fragments are produced in N–Cα bond cleavage. Because of the sporadic nature of radical a-ions in MS/MS spectra, a large MS/MS database is needed for obtaining reliable statistics [5, 6]. We created and interrogated such a database, testing the origin of a-ions in ECD. They indeed arise from the process (1), which gives complementary \( a \cdot \) and y' fragments. Intriguingly, the most abundant \( a \cdot \)/y' pairs are found to come from the cleavage of the same backbone link as the most abundant c' and \( z \cdot \) complementary ions. This result gives strong albeit indirect support to the “local” ECD mechanism, in which the N–Cα bond dissociation occurs at the site of charge solvation . However, a second, almost equally strong peak is observed in the c'/\( z \cdot \) fragment distribution four residues away from the \( a \cdot \)/y' cleavage, which supports the indirect mechanism [7, 8, 9, 10]. Below, we describe these results and their meaning for the ECD mechanism.
The importance of this finding is in the first direct confirmation of a “local” ECD mechanism, albeit the one related to a different backbone bond than the N–Cα bond. The local mechanism postulates the electron capture directly onto the charged group, and a bond cleavage next to that group .
In contrast, the non-local mechanisms [7, 8, 9] assume electron capture on a non-charged atom near, but not immediately at, the charge location. One of the objections to a local mechanism is that direct electron-proton recombination releases too much energy to be accommodated by the system on the time scale of electronic transitions, which enhances the probability of the reversed reaction–electron release to the continuum . However, if (1) is validated, then this objection is lifted.
It is possible to estimate the relative contributions of the local versus non-local mechanism in N–Cα bond cleavage. To this end, only MS/MS spectra were selected in which both c'/\( z \cdot \) and \( a \cdot \)/y' complementary pairs were present. The local model predicts that the most abundant c'/\( z \cdot \) and \( a \cdot \)/y' complementary pairs will come from the cleavage of the same backbone link.
The authors acknowledge support for this by the Swedish Research Council and KAW Foundation. D.M.G. is grateful for support from a Wenner-Gren postdoctoral fellowship.
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