General Mechanism of C_α-C Peptide Backbone Bond Cleavage in Matrix-Assisted Laser Desorption/Ionization In-Source...

Nitrogen-centered and β-carbon-centered hydrogen-deficient peptide radicals are considered to be intermediates in the matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD)-induced C_α-C bond cleavage of peptide backbones when using an oxidizing matrix. To understand the general mechanism of C_α-C bond cleavage by MALDI-ISD, I study the fragmentation of model peptides and investigate the fragment formation pathways using calculations with density functional theory and transition state theory. The calculations indicate that the nitrogen-centered radical immediately undergoes C_α-C bond cleavage, leading to the formation of an a•/x fragment pair. In contrast, the dissociation of the β-carbon-centered radical is kinetically feasible under MALDI-ISD conditions, leading to the formation of an a/x• fragment pair. To discriminate these processes, I focus on the yield of d fragments, which originate from a• radicals through radical-induced side-chain loss, not from a fragments. The C_α-C bond cleavage on the C-terminal side of the carbamidomethylated cysteine residue is found to produce d fragments instead of a fragments. According to the calculation of the rate constant, the corresponding fragmentation occurs within 1 ns. The intense signal arising from d fragments and the lack of or weak signal from a fragments strongly suggest that the C_α-C bond cleavage occurs through a nitrogen-centered radical intermediate. In addition to the side-chain loss, the resulting a• radical undergoes hydrogen atom abstraction by the matrix. The results for a deuterium-labeled peptide indicate that the matrix abstracts a hydrogen atom from either the amide nitrogen or the β-carbon.