Identification of Amide Oxygen as First Protonation Site in Gaseous Short Peptides: Theoretical Evidence from a First-Principles Study

Although previous IRMPD (infrared multiple photon dissociation) experiments proposed the presence of protonation of the C=O peptide bond in certain tripeptides with lower energy than traditional amino protonation, subsequent theoretical calculations have revealed that their conclusions are unreliable. Therefore, it has become imperative to explore the existence of such molecules. In this study, based on reasonable speculation, four dipeptides and four tripeptides were selected and their protonated configurations were systematically searched. High-level theoretical calculations using the composite CBS-QB3 method indicated that at least two dipeptides, GP and GV (G: glycine, P: proline, V: valine), have been identified as the candidate molecules with protonation at the amide oxygen as the global minimum. GP is also identified as the smallest dipeptide with the cis-peptide conformation as the global minimum. The electrostatic potentials and the transition states between the two protonated forms have been calculated to uncover the determining mechanism for the predominance of peptide bond protonation. In addition, the chemical (infrared, IR) and electronic (X-ray photoelectronic spectroscopy and near-edge X-ray absorption fine structures, XPS and NEXAFS) structural calculations were performed to distinguish between these different protonated forms in future experiments. This study provides valuable insights into the competitive coexistence between the two protonated forms of short peptides and deepens our understanding of the protonation process in the early stage of protein synthesis.