Shin Nah-Young, Liu Qinfeng, Stamer Sheryl L, Liebler Daniel C
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Chem Res Toxicol. 2007 Jun;20(6):859-67. doi: 10.1021/tx700031r. Epub 2007 May 5.
Liver microsomes are widely used to study xenobiotic metabolism in vitro, and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible, and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB- but not BMCC-induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.
肝微粒体被广泛用于体外研究外源化合物代谢，与微粒体蛋白的共价结合作为反应性代谢物介导毒性的替代标志物。我们应用液相色谱 - 串联质谱法（LC-MS-MS）来鉴定人肝微粒体中生物素标记的模型亲电试剂1-生物素酰胺基-4-（4'-[马来酰亚胺基乙基环己烷]-羧酰胺基）丁烷（BMCC）和N-碘乙酰基-N-生物素基己二胺（IAB）的蛋白质靶点。凝胶内胰蛋白酶消化产生的生物素标记肽通过生物素 - 抗生物素蛋白色谱法富集，并用LC-MS-MS鉴定263种蛋白质中BMCC和IAB的376个微粒体半胱氨酸硫醇靶点。蛋白质加合物具有选择性和可重复性，两种亲电试剂仅使70种蛋白质中的90个特定半胱氨酸位点（约占总数的25％）发生加合。加合选择性的差异与化合物的不同生物学效应相关，因为IAB而非BMCC在HEK293细胞中诱导内质网应激。对微粒体谷胱甘肽 - S - 转移酶半胱氨酸50（IAB和BMCC的共同靶点）进行靶向LC-MS-MS分析，检测到微粒体孵育期间反应性对乙酰氨基酚代谢物N-乙酰 - 对苯醌亚胺的时间依赖性加合。结果表明，亲电试剂选择性地与微粒体蛋白加合，但表现出不同的靶点选择性，这与毒性差异相关。因此，与放射性标记化合物的大量共价结合相比，对选定的微粒体蛋白加合反应进行分析可以提供更具体的潜在毒性指示。
