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

Abstract

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) causes the selective cleavage of C α -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 C α -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 C α -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 C α -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 C α -C bond cleavage at the Cterminal 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, whenthe 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.

Ultraviolet-Laser-Induced Electron Transfer from Peptides to an Oxidizing Matrix: Study of the First Step of MALDI In-Source Decay Mass Spectrometry

“…Note that the yield of the [d] + fragments is dependent on the dissociation rate constant of the corresponding [a]• radicals. 28 The results strongly support the proposed mechanism of C α −C bond cleavage through the formation of an [a]•/[x − H] fragment pair, as shown in Scheme 1a.…”

“…To investigate the possible sites for hydrogen abstraction, the dissociation energies for C–H and N–H bonds in Ala–Ala–Ala­(Me) ([M] in Figure a) were calculated, and these values are summarized in Table . According to previous reports, MALDI-ISD does not provide the β-carbon-centered peptide radical intermediate. , In other word, the hydrogen abstraction from the β-carbon atom in peptide does not occur during the MALDI-ISD process. , In consequence, the abstraction of hydrogen from the N -methyl carbon atoms in Ala–Ala–Ala­(Me) was confirmed to occur at a lower energy than that required for the abstraction of hydrogen from the β-carbon atom. In contrast, the energy required for hydrogen abstraction from the α-carbon atom is lower than that from the N -methyl carbon.…”

“…According to previous reports, MALDI-ISD does not provide the β-carbon-centered peptide radical intermediate. 27,28 In other word, the hydrogen abstraction from the β-carbon atom in peptide does not occur during the MALDI-ISD process. 27,28 In consequence, the abstraction of hydrogen from the N-methyl carbon atoms in Ala−Ala−Ala(Me) was confirmed to occur at a lower energy than that required for the abstraction of hydrogen from the βcarbon atom.…”

“…Consequently, peptide and protein sequencing by MALDI-ISD with an oxidizing matrix is generally performed by interpreting the mass differences between series of consecutive [a] + fragments . An exception is the cleavage of C α –C bonds at the C-terminal side of cysteine (Cys) and derivative residues, which produce intense [d] + rather than [a] + signals. , The intense [d] + signals originating from the bond cleavage at the C-terminal side of Cys and derivative residues can be explained by the high dissociation rate constant of the corresponding [a]· radicals. Note that the yield of the [d] + fragments is dependent on the dissociation rate constant of the corresponding [a]· radicals .…”

“…An exception is the cleavage of C α –C bonds at the C-terminal side of cysteine (Cys) and derivative residues, which produce intense [d] + rather than [a] + signals. , The intense [d] + signals originating from the bond cleavage at the C-terminal side of Cys and derivative residues can be explained by the high dissociation rate constant of the corresponding [a]· radicals. Note that the yield of the [d] + fragments is dependent on the dissociation rate constant of the corresponding [a]· radicals . The results strongly support the proposed mechanism of C α –C bond cleavage through the formation of an [a]·/[x – H] fragment pair, as shown in Scheme a.…”