Investigation of the mechanism of electron capture and electron transfer dissociation of peptides with a covalently attached free radical hydrogen atom scavenger

The mechanisms of electron capture and electron transfer dissociation (ECD and ETD) are investigated by covalently attaching a free-radical hydrogen atom scavenger to a peptide. The 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) radical was chosen as the scavenger due to its high hydrogen atom affinity (ca. 280 kJ/mol) and low electron affinity (ca. 0.45 eV), and was derivatized to the model peptide, FQX^TEMPOEEQQQTEDELQDK. The X^TEMPO residue represents a cysteinyl residue derivatized with an acetamido-TEMPO group. The acetamide group without TEMPO was also examined as a control. The gas phase proton affinity (882 kJ/mol) of TEMPO is similar to backbone amide carbonyls (889 kJ/mol), minimizing perturbation to internal solvation and sites of protonation of the derivatized peptides. Collision-induced dissociation (CID) of the TEMPO-tagged peptide dication generated stable odd-electron b and y type ions without indication of any TEMPO radical induced fragmentation initiated by hydrogen abstraction. The type and abundance of fragment ions observed in the CID spectra of the TEMPO and acetamide tagged peptides are very similar. However, ECD of the TEMPO-labeled peptide dication yielded no backbone cleavage. We propose that a labile hydrogen atom in the charge reduced radical ions is scavenged by the TEMPO radical moiety, resulting in inhibition of N-C_α backbone cleavage processes. Supplemental activation after electron attachment (ETcaD) and CID of the charge-reduced precursor ion generated by electron transfer of the TEMPO-tagged peptide dication produced a series of b + H (b^H) and y + H (y^H) ions along with some c ions having suppressed intensities, consistent with stable O-H bond formation at the TEMPO group. In summary, the results indicate that ECD and ETD backbone cleavage processes are inhibited by scavenging of a labile hydrogen atom by the localized TEMPO radical moiety. This observation supports the conjecture that ECD and ETD processes involve long-lived intermediates formed by electron capture/transfer in which a labile hydrogen atom is present and plays a key role with low energy processes leading to c and z ion formation. Ab initio and density functional calculations are performed to support our conclusion, which depends most importantly on the proton affinity, electron affinity and hydrogen atom affinity of the TEMPO moiety.