Specific Cα-C Bond Cleavage of β-Carbon-Centered Radical Peptides Produced by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

Radical-driven dissociation (RDD) of hydrogen-deficient peptide ions [M - H + H] has been examined using matrix-assisted laser dissociation/ionization in-source decay mass spectrometry (MALDI-ISD MS) with the hydrogen-abstracting matrices 4-nitro-1-naphthol (4,1-NNL) and 5-nitrosalicylic acid (5-NSA). The preferential fragment ions observed in the ISD spectra include N-terminal [a] + ions and C-terminal [x], [y + 2], and [w] ions which imply that β-carbon (Cβ)-centered radical peptide ions [M - Hβ + H] are predominantly produced in MALDI conditions. RDD reactions from the peptide ions [M - Hβ + H] successfully explains the fact that both [a] and [x] ions arising from cleavage at the Cα-C bond of the backbone of Gly-Xxx residues are missing from the ISD spectra. Furthermore, the formation of [a] ions originating from the cleavage of Cα-C bond of deuterated Ala(d3)-Xxx residues indicates that the [a] ions are produced from the peptide ions [M - Hβ + H] generated by deuteron-abstraction from Ala(d3) residues. It is suggested that from the standpoint of hydrogen abstraction via direct interactions between the nitro group of matrix and hydrogen of peptides, the generation of the peptide radical ions [M - Hβ + H] is more favorable than that of the α-carbon (Cα)-centered radical ions [M - Hα + H] and the amide nitrogen-centered radical ions [M - H + H], while ab initio calculations indicate that the formation of [M - Hα + H] is energetically most favorable. Graphical Abstract ᅟ.

Matrix‐dependent a/x pair and overdegraded w/y/z ions generated by radical‐directed dissociation of peptide radical cations [M]+ in matrix‐assisted laser desorption/ionization in‐source decay

“…[11][12][13] It is known that hydrogen- according to the sites of charge and the presence of specific residues. [5][6][7][8]14,15 For a rational explanation of the RDD of peptide radical cations [M] .+ , the site of the radical (unpaired electron) is critical because the formation of fragment ions is governed by the radical site. 8,[14][15][16] Here, we examine the fragment ions depending on a matrix in MALDI-ISD experiments with hydrogen-abstracting matrix.…”

“…[5][6][7][8]14,15 For a rational explanation of the RDD of peptide radical cations [M] .+ , the site of the radical (unpaired electron) is critical because the formation of fragment ions is governed by the radical site. 8,[14][15][16] Here, we examine the fragment ions depending on a matrix in MALDI-ISD experiments with hydrogen-abstracting matrix. In order to observe amino (N)-and carboxyl (C)-terminal characteristic fragments, model peptides containing an arginine (Arg) residue at the Nand C-terminal sides were used.…”

“…It is known that hydrogen‐deficient radical cations [M] . + of peptides result in characteristic fragment ions such as [ a ] + , [ b ] + , [ d ] + , [ w ] + , [ x ] + , [ y ] + , and [ z ] + ions according to the sites of charge and the presence of specific residues 5–8,14,15 . For a rational explanation of the RDD of peptide radical cations [M] .…”

“…For a rational explanation of the RDD of peptide radical cations [M] . + , the site of the radical (unpaired electron) is critical because the formation of fragment ions is governed by the radical site 8,14–16 …”

“…When 3,5-DNSA matrix was used, the ISD spectra of both peptides showed preferential [x] + ions and some minor [y] + , [w] + , and/or [z] + ions. The preferential [x] + ions are formed as the pair of [a] + ions 14,15. As already reported,8 the ISD spectra of the amino (N)-terminal Arg containing peptides LRAGly14 and RGV-d5 mainly showed [a] + ions originating from cleavage at the Cα-C bond of the peptide backbone when 3,5-DNSA was used, whereas the use of 4,1-NNL resulted in major [a] + ions and some [d] + ions.…”