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 (REvert) for 1a(+) and 2a(+). The recombination energies, REvert = 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(center dot) 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(center dot) was calculated to spontaneously isomerize upon electron capture to a stable dihydroxycarbinyl isomer (2e(center dot)) that can undergo consecutive and competitive isomerizations by proline ring opening and intramolecular hydrogen atom transfers to yield stable radical isomers. Radical 2e(center dot) 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-alpha 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-alpha bonds adjacent to proline residues in tryptic peptides and also for some peculiar behavior of proline-containing protein cation-radicals.