The formation and radical-directed dissociation of multiple hydrogen-abstracted peptide cations [M + H − mH]• + has been reported using MALDI-ISD with dinitro-substituted matrices. The MALDI-ISD of synthetic peptides using 3,5dinitrosalicylic acid (3,5-DNSA) and 3,4-dinitrobenzoic acid (3,4-DNBA) as matrices resulted in multiple hydrogen abstraction from the analyte [M + H] + and fragment [a] + ions, i.e., [M + H − mH] + and [a − mH] + (m = 1−8). All of the ISD spectra showed unusually intense [a] + ions originating from cleavage at the Cα−C bond of the Leu-Xxx residues when peptides without Phe/Tyr/ His/Cys residues were used. The intensity of the [a n ] + series ions generated using 3,5-DNSA and 3,4-DNBA rapidly decreased with increasing residue number n, suggesting cleavage at multiradical sites of [M + H − mH] •+ . It was suggested that multiple hydrogen abstraction from protonated peptides [M + H] + mainly takes place from the backbone amide nitrogen.

Fragmentation of peptide radical cations [M].+ has been examined using matrix‐assisted laser desorption/ionization (MALDI) in‐source decay (ISD) with hydrogen‐abstracting nitro‐substituted matrices. The ISD spectra of peptides containing an arginine (Arg) residue at carboxyl (C)‐termini showed preferential [w]+ ions when 4‐nitro‐1‐naphthol (4,1‐NNL) matrix was used, whereas the use of 3,5‐dinitrosalicylic acid (3,5‐DNSA) resulted in preferential [x]+ ions. Minor or some [d]+, [x]+, [y]+, and [z]+ ions were also observed. For peptides containing Arg residue at amino (N)‐termini, the ISD spectra showed preferential [a]+ ions independent of matrix used. The observed [a]+, [w]+, [x]+, [y]+, and [z]+ ions can be rationally explained by radical‐directed dissociation (RDD) of the peptide radical cations [M].+, although [d]+ ions may be formed via Norrish Type I cleavage and/or by RDD of [M].+ ions. The formation of overdegraded [d]+, [w]+, [y]+, and [z]+ ions is discussed from the standpoint of the internal energy of radical cations [M].+ and radical fragment ions [a + H].+ and [x + H].+ deposited via collisional interactions with excited matrix molecules in the MALDI plume. The radical site of the peptide cations [M].+ was presumed to be backbone amide nitrogen, from MALDI‐ISD data with three different deuterated amino acids.

e formation and radical-directed dissociation RDD of multiple hydrogen abstracted peptide cations M H-mH • have been examined using MALDI In-source decay ISD with four di erent dinitro-substituted matrices, i.e., 3,5-dinitrosalicylic acid 3,5-DNSA , 3,4-dinitrobenzoic acid 3,4-DNBA , 1,5-dinitronaphthalene 1,5-DNN and 2,4-dinitro-1-naphthol 2,4-DNNL. e MALDI-ISD of peptides using 3,5-DNSA and 3,4-DNBA as matrices resulted in multiple hydrogen abstraction from protonated peptide M H and production of fragment a ions, i.e., M H-mH • and a-mH • m 1 6. Fragmentation pattern of the intensity of a n ions with 4-nitro-1-naphthol 4,1-NNL depended on amino acid residue, while that of the a n ions with 3,5-DNSA did not.

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