Dissecting the Proline Effect: Dissociations of Proline Radicals Formed by Electron Transfer to Protonated Pro-Gly and Gly-Pro Dipeptides in the Gas Phase

Abstract

We report a combined experimental and computational study of the proline effect in model dipeptides Pro-Gly and Gly-Pro. Gas-phase protonated peptide ions were discharged by glancing collisions with potassium or cesium atoms at 3 keV collision energies, and the peptide radical intermediates and their dissociation products were analyzed following collisional ionization to anions. The charge reversal (⁺CR^-) mass spectra of (Pro-Gly + H)⁺ (1a⁺) and (Gly-Pro + H)⁺ (2a⁺) showed dramatic differences and thus provided a sensitive probe of ion structure. Whereas 1a⁺ completely dissociated upon charge inversion, 2a⁺ gave a nondissociated anion as the most abundant product. Ab initio and density functional theory calculations provided structures and vertical recombination energies (RE_vert) for 1a⁺ and 2a⁺. The recombination energies, RE_vert = 3.07 and 3.36 eV for 1a⁺ and 2a⁺, respectively, were lower than the alkali metal ionization energies and indicated that the collisional electron transfer to the peptide ions was endoergic. Radical 1a^• was found to exist in a very shallow local energy minimum, with transition state energies for loss and migration of H indicating very facile dissociation. In contrast, radical 2a^• was calculated to spontaneously isomerize upon electron capture to a stable dihydroxycarbinyl isomer (2e^•) that can undergo consecutive and competitive isomerizations by proline ring opening and intramolecular hydrogen atom transfers to yield stable radical isomers. Radical 2e^• and its stable isomers were calculated to have substantial electron affinities and thus can form the stable anions that were observed in the ⁺CR^- mass spectra. The calculated TS energies and RRKM kinetic analysis indicated that peptide N−C_α bond dissociations compete with pyrrolidine ring openings triggered by radical sites at both the N-terminal and C-terminal sides of the proline residue. Open-ring intermediates were found in which loss of an H atom was energetically preferred over backbone dissociations. This provided an explanation for the proline effect causing low incidence of electron capture dissociations of N−C_α bonds adjacent to proline residues in tryptic peptides and also for some peculiar behavior of proline-containing protein cation-radicals.