Cooperative dissociation of peptide backbones and side-chains during matrix-assisted laser desorption/ionization in-source decay mediated by hydrogen abstraction.

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) causes the selective cleavage of Cα -C peptide bonds when an oxidizing matrix is used, and the fragmentation involves the hydrogen abstraction from a peptide by a matrix. The hydrogen abstraction from either an amide nitrogen or β-carbon atom has been proposed to be the initial step leading to the Cα -C bond cleavage. In this regard, the production of [a]+ fragments originated upon bond cleavage at the C-terminal side of phenylglycine residues strongly suggested that that the Cα -C bond cleavage occurred through a nitrogen-centered radical intermediate and that the fragmentation through a β-carbon-centered radical intermediate can be ruled out from the MALDI-ISD process, because phenylglycine residues do not contain β-carbon atoms. The Cα -C bond cleavage of such nitrogen-centered radical initially produced an [a]•/[x - H] fragment pair, and then the [a]• radical either reacted with the matrix or underwent loss of the side-chain, leading to [a - H] or [d - H] fragment. The Cα -C bond cleavage at the C-terminal side of phenylglycine and phenylalanine residues only generated [a]+ fragments, whereas that of homophenylalanine and S-methylated cysteine residues provided both [a]+ and [d]+ fragments. The yield of [d]+ fragments was dependent on the chemical stability of the resultant radicals formed upon side-chain loss. MALDI-ISD produced [M - H + matrix]+ , [M - 16 + H]+ , [M - 32 + H]+ , and [d]+ fragments, when the analyte peptide contained a methionine residue. These fragments were formed upon abstraction of a hydrogen atom from the side-chain of a methionine residue and its subsequent reaction with the matrix. The oxidation of methionine residues suppressed the hydrogen abstraction from their side-chain.