![]() Regarding the mechanism, hydrogen abstraction from the peptide by the matrix produces a hydrogen-deficient peptide radical intermediate, which undergoes C α–C bond cleavage. More recently, hydroxy-nitrobenzoic acid isomers, 7,7,8,8-tetracyanoquinodimethane derivatives, and 4-nitro-1-naphthol (4,1-NNL) were reported to efficiently produce fragment ions due to a C α–C bond cleavage. MALDI-ISD with 5-NSA permits identification of phosphorylated and isoaspartate residues in peptides. In contrast to the N–C α bond cleavage, the cleavage of the C α–C bond was recently found to occur when 5-nitrosalicylic acid (5-NSA) was used as an oxidizing matrix for MALDI-ISD. Consequently, MALDI-ISD is becoming increasingly important in the field of proteomics, and this is likely to continue. Moreover, MALDI-ISD preferentially produces c′ and z′ fragments without degradation of labile post-translational modifications, so the locations of phosphorylation, O-glycosylation, and polyethylene glycosylation in proteins are determined. Additionally, a MALDI-ISD–based pseudo-MS 3 method, that is mass selection of ISD ions followed by fragmentation with post-source decay, provides information on the identity of the C- and N-terminus of the protein and enables direct identification of a target protein in a mixture. To obtain accurate mass differences, MALDI-ISD employing a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was recently developed. Because z′ fragments often appear as intense peaks, protein sequencing by MALDI-ISD is performed by interpreting mass differences between series of consecutive c′ and z′ ions. fragment either reacts with the matrix or undergoes further fragmentation to give various fragments such as, z′, z, w, and.The resulting aminoketyl radical intermediate eventually undergoes N–C α bond cleavage, leading to the formation of a c′/ z MALDI-ISD is initiated by laser-induced hydrogen radical transfer from the matrix to the analyte peptide, which has been suggested to occur via hydrogen bonding between the peptide and the matrix prior to desorption. In particular, the most impressive of these matrices is 1,5-DAN, which efficiently produces fragment ions and allows rapid characterization of the sequence of intact proteins. Furthermore, the use of specific reducing matrices such as 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, and 1,5-diaminonaphthalene (1,5-DAN) can cause in-source decay (ISD) during MALDI experiments. An important advantage of MALDI is that intact peptide ions can be produced without abundant fragmentation, which facilitates rapid characterization of a protein digest. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a major analytical tool for the characterization of proteins. The results for a deuterium-labeled peptide indicate that the matrix abstracts a hydrogen atom from either the amide nitrogen or the β-carbon. radical undergoes hydrogen atom abstraction by the matrix.In addition to the side-chain loss, the resulting a 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. According to the calculation of the rate constant, the corresponding fragmentation occurs within 1 ns. 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. radicals through radical-induced side-chain loss, not from a fragments.To discriminate these processes, I focus on the yield of d fragments, which originate from a 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 The calculations indicate that the nitrogen-centered radical immediately undergoes C α–C bond cleavage, leading to the formation of an a 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. 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. ![]()
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