Alkylation of Cytochrome c by (Glutathion-S-yl)-1,4-benzoquinone and Iodoacetamide Demonstrates Compound-Dependent Site Specificity
详细信息 查看全文
- 作者：Maria D. Person ; Daniel E. Mason ; Daniel C. Liebler ; Terrence J. Monks ; and Serrine S. Lau
- 刊名：Chemical Research in Toxicology
- 出版年：2005
- 出版时间：January 2005
- 年：2005
- 卷：18
- 期：1
- 页码：41 - 50
- 全文大小：247K
- 年卷期：v.18,no.1(January 2005)
- ISSN：1520-5010
文摘
The reaction of cytochrome c with the electrophilic compounds (glutathion-S-yl)-1,4-benzoquinone (GSBQ) and iodoacetamide was studied using mass spectrometry. GSBQ is anephrotoxic quinol-thioether metabolite of benzoquinone, while iodoacetamide is an alkylatingagent targeting cysteine thiols. Both chemicals formed covalent adducts with cytochrome c.GSBQ formed adducts with cytochrome c at pH 6 on several histidine and lysine residues. Ata pH >7, the initial product rearranged to a disubstituted cyclic quinone species preferentiallyfound at two sites on the protein, Lys25-Lys27 and Lys86-Lys87, via quinol amine linkages.These two sites were previously determined to be the targets of benzoquinone adduct formation[Person et al. (2003) Chem. Res. Toxicol. 16, 598-608]. Cyclic reaction products arepreferentially formed at two sites on the protein because of the presence of multiple basicresidues in a conformationally flexible region whereas noncyclic products bind to a broadspectrum of available lysine and histidine nucleophiles. Iodoacetamide was a less selectivealkylating agent able to form adducts on the majority of the nucleophilic sites of the protein.MS/MS spectra were used to identify signature ions for GSBQ-adducted peptides from thecharacteristic fragmentation patterns. Neutral losses of the 129 Da 
-glutamate residue andof the 273 Da glutathione moiety were found in both cysteine thiol- and lysine amine-linkedGSBQ adduct MS/MS. Characteristic fragment ions were used in conjunction with the scoringalgorithm for spectral analysis to search for adducted species present at low levels in the sample,and the analysis is applicable generally to detection of glutathione conjugates by MS/MS.Parallel analysis using matrix-assisted laser desorption/ionization-MS to compare spectra ofcontrol and treated samples allowed identification of peptide adducts formed by direct additionof GSBQ and by the subsequent loss of the glutathione moiety in a pH-dependent cyclizationreaction.
-glutamate residue andof the 273 Da glutathione moiety were found in both cysteine thiol- and lysine amine-linkedGSBQ adduct MS/MS. Characteristic fragment ions were used in conjunction with the scoringalgorithm for spectral analysis to search for adducted species present at low levels in the sample,and the analysis is applicable generally to detection of glutathione conjugates by MS/MS.Parallel analysis using matrix-assisted laser desorption/ionization-MS to compare spectra ofcontrol and treated samples allowed identification of peptide adducts formed by direct additionof GSBQ and by the subsequent loss of the glutathione moiety in a pH-dependent cyclizationreaction.