Protein targets of reactive electrophiles in human liver microsomes
Protein targets of reactive electrophiles in human liver microsomes
复制标题
DOI：
10.1021/tx700031r
复制
发表时间：
2007-06-01
影响因子：
4.1
通讯作者：
Liebler, Daniel C.
中科院分区：
文献类型：
--
作者：
Shin, Nah-Young;Liu, Qinfeng;Liebler, Daniel C.
肝微粒体被广泛用于体外研究外源代谢，与微粒体蛋白的共价结合可作为反应性代谢物介导毒性的替代标志物。采用液相色谱-串联质谱法（LC-MS-MS）鉴定了人肝微粒体中生物素标记的模型亲电试剂1-生物酰胺-4-（4′-[马来酰亚胺乙基环己烷]-羧胺）丁烷（BMCC）和n-碘乙酰- n-生物素基己二胺（IAB）的蛋白靶点。用生物素-亲和素层析富集凝胶内胰蛋白酶消化得到的生物素标记肽，并用LC-MS-MS鉴定了263个蛋白中的376个微粒体BMCC和IAB的半胱氨酸硫醇靶点。蛋白质内聚具有选择性和可重复性，70种蛋白质中只有90个特定的半胱氨酸位点（约占总数的25%）被两种亲电试剂内聚。内合选择性的差异与化合物的不同生物学效应相关，如IAB-而非bmcc诱导HEK293细胞内质网应激。靶向LC-MS-MS分析微粒体谷胱甘肽- s -转移酶半胱氨酸50 （IAB和BMCC的靶标），检测到在微粒体培养过程中，活性对乙酰氨基酚代谢物n -乙酰基-对苯醌亚胺的时间依赖性内合。结果表明，亲电试剂选择性地加合微粒体蛋白，但表现出不同的目标选择性，这与毒性的差异有关。因此，与放射性标记化合物的大量共价结合相比，分析选定的微粒体蛋白内合反应可以提供更具体的潜在毒性指示。
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.