Theoretical investigation for the proline effect on peptide fragmentation induced by matrix-assisted laser desorption/ionization in-source decay with an oxidizing matrix

The use of an oxidizing matrix for matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) specifically induces the cleavage of Cα-C bonds on the peptide backbone, except for bonds located on the N-terminal side of proline residues. To examine the effect of Pro residues on bond cleavage induced by MALDI-ISD with an oxidizing matrix, the small dipeptides AcAA-NH₂ and AcAP-NH₂ were used as models. As the fragmentation is initiated by electron transfer from the peptide to the oxidizing matrix, the dissociation chemistry of the cation radical forms of the model peptides [AcAA-NH₂]⁺• and [AcAP-NH₂]⁺• was investigated using quantum chemistry calculations. The [AcAA-NH₂]⁺• can produce fragment ions not only due to Cα–C bond cleavage, but also peptide bond cleavage. Quantum chemistry calculations indicated that peptide bond cleavage of [AcAA-NH₂]⁺• occurs more slowly compared to Cα–C bond cleavage. Instead of Cα–C bond cleavage, the bond on the N-terminal side of Pro residue undergoes peptide bond cleavage during MALDI-ISD with an oxidizing matrix, due to the lack of an amide hydrogen in the Pro residue. The [AcAP-NH₂]⁺• undergoes proton migration from the δ-carbon of the Pro residue. Depending on the proton binding site in the peptide cation radical, the peptide bond cleavage of [AcAP-NH₂]⁺• results in the formation of either [b₁]⁺, and [y₁]•, or [a₁]• and [y₁]⁺. These theoretical results are consistent with experimental findings, and the newly proposed mechanism involving peptide cation radical formation followed by proton migration provides a more accurate model for the MALDI-ISD processes.