Studies on the clinical proteomics with bile acid tagged proteins as the targeted molecules

形成共价结合的蛋白质加合物与石胆酸（LCA）已被提出作为一个可能的解释，这种胆汁酸的超敏反应和毒性反应。因此，我们使用对LCA的3α-羟基-5 β-类固醇部分具有高亲和力的抗体，通过免疫沉淀在胆管结扎的大鼠肝脏中捕获LCA修饰的细胞蛋白。通过结合使用MALDI-TOFMS和计算机辅助程序的2-DE基本蛋白质组学技术对捕获的蛋白质进行结构分析的结果表明存在LCA结合的Rab蛋白，进一步研究了谷胱甘肽（GSH）与活性酰基-已经进行了胆汁酸的连接代谢物以证明解毒机制的存在。为此，化学合成了五种主要胆汁酸的真正的GSH缀合物 ...更多信息 并通过电喷雾串联质谱在正负离子模式下对它们的结构进行了表征。孵育胆酰-腺苷酸和胆酰-CoA，胆酸（CA）的反应性代谢产物，与GSH在磷酸盐缓冲液（pH 7.5）的解决方案表明，这两种化合物能够在体外酰化GSH。此外，我们克隆并表达了大鼠和人肝脏胆汁酸CoA连接酶，以进一步了解酰基腺苷酸和胆汁酸结合蛋白的形成机制。最后，我们的工作是通过在LCA的C-3和C-24位引入桥，将LCA共价结合到活化的琼脂糖上，利用亲和吸附剂，在大鼠肝脏中寻找与LCA具有亲和性的蛋白。在通过SDS-PAGE分离后对捕获的蛋白质进行LC/ESI-MS分析的结果揭示了功能蛋白质的存在，即，氨甲酰磷酸合酶I、谷氨酸脱氢酶、酰基辅酶A酰基转移酶、谷胱甘肽-S-转移酶和过氧化氢酶，表明LCA抑制这些酶导致肝毒性出现的假设。少

Formation of covalently bound protein adducts with lithocholic acid (LCA) has been proposed as a possible explanation for hypersensitivity and toxic responses of this bile acid. We therefore carried out to capture of LCA-modified cellular proteins in the bile duct-ligated rat liver by immunoprecipitation using an antibody with high affinity to the 3α-hydroxy-5β-steroid moiety of LCA. Results of the structural analysis of the captured proteins by the basic proteomic technique of 2-DE combined with the use of MALDI-TOFMS and computer assisted programs indicated the presence of LCA bound Rab proteins, which may be the course of LCA-induced liver toxicity.Further studies on the formation of the glutathione (GSH) conjugates via trans-acylation reactions between GSH and reactive acyl-linked metabolites of bile acids have been carried out to demonstrate the presence of detoxification mechanism. For this purpose, the authentic GSH conjugates of five major bile acids were chemically synthesized … More and their structures were characterized by ESI-tandem mass spectrometry in negative and positive ion modes. Incubation of cholyl-adenylate and cholyl-CoA, reactive metabolites of cholic acid (CA), with GSH in a phosphate buffer (pH 7.5) solution demonstrated that both compounds are able to acylate GSH in vitro. In addition, we cloned and expressed rat and human liver bile acid CoA-ligase to better understand the mechanism for the formation of acyl-adenylate and bile acid bound proteins.Finally, our efforts were directed to explore the proteins having affinity with LCA in rat liver by using affinity adsorbents, in which LCA was covalently bound to an activated agarose through the bridges introduced at the C-3 and C-24 positions of LCA. Results of LC/ESI-MS^n analysis of the captured proteins after separation by SDS-PAGE revealed the presence of functional proteins, i.e., carbamoyl phosphate synthase I, glutamate dehydrogenase, acyl-CoA acyltransferase, glutathione-S-transferase, and catalase, indicating the hypothesis involved in the appearance of the liver toxicity by inhibition of these enzymes by LCA. Less