Cytosolic and nuclear protein targets of thiol-reactive electrophiles
Cytosolic and nuclear protein targets of thiol-reactive electrophiles
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DOI：
10.1021/tx050312l
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发表时间：
2006-01-01
影响因子：
4.1
通讯作者：
Liebler, DC
中科院分区：
文献类型：
--
作者：
Dennehy, MK;Richards, KAM;Liebler, DC
由有毒药物和化学品形成的反应性亲电体和内源性氧化应激共价修饰蛋白质。虽然蛋白质共价结合被认为可以引发各种适应性和毒性反应，但蛋白质靶点的身份通常是未知的，因为蛋白质结构特征赋予了对修饰的敏感性。我们分析了HEK 293细胞核和细胞质蛋白质组中的蛋白质靶点，这些蛋白质组在体外用两种生物素标记的硫醇反应性亲电试剂（+）-生物素基-碘乙酰胺基-3，6-二氧杂辛二胺（PEO-IAB）和1-生物素酰胺基-4-（4 '-[马来酰亚胺乙基环己烷]-甲酰氨基）丁烷（BMCC）处理。生物素化的肽被捕获的亲和富集使用中性亲和素珠，并加合物的肽，然后通过多维液相色谱-串联质谱分析。共897加合物被映射到539个蛋白质中的不同半胱氨酸残基。加合是选择性和可重复的，并且> 90%的所有加合蛋白质仅在一个或两个位点处被修饰。125个半胱氨酸（占总数的14%）的核心组始终由两种亲电试剂修饰。几种蛋白质结构域结构和基序的选择性修饰表明某些蛋白质家族对烷基化特别敏感。这种方法可以扩展到其他蛋白质损伤的氧化剂和亲电体的研究，并可以提供新的见解毒性和疾病的蛋白质损伤的目标和后果。
Reactive electrophiles formed from toxic drugs and chemicals and by endogenous oxidative stress covalently modify proteins. Although protein covalent binding is thought to initiate a variety of adaptive and toxic responses, the identities of the protein targets are generally unknown, as are protein structural features that confer susceptibility to modification. We have analyzed the protein targets in nuclear and cytoplasmic proteomes from HEK293 cells treated in vitro with two biotin-tagged, thiol-reactive electrophiles, (+)-biotinyl-iodoacetamidyl-3, 6-dioxaoctanediamine (PEO-IAB) and 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC). Biotinylated peptides were captured by affinity enrichment using neutravidin beads, and the adducted peptides were then analyzed by multidimensional liquid chromatography-tandem mass spectrometry. A total of 897 adducts were mapped to different cysteine residues in 539 proteins. Adduction was selective and reproducible, and > 90% of all adducted proteins were modified at only one or two sites. A core group of 125 cysteines (14% of the total) was consistently modified by both electrophiles. Selective modification of several protein domain structures and motifs indicates that certain protein families are particularly susceptible to alkylation. This approach can be extended to studies of other protein-damaging oxidants and electrophiles and can provide new insights into targets and consequences of protein damage in toxicity and disease.