一维蛋白质免疫印迹结合液相色谱-串联质谱分析以鉴定大鼠尿液中的化学加合蛋白。
One-dimensional western blotting coupled to LC-MS/MS analysis to identify chemical-adducted proteins in rat urine.
作者信息
Labenski Matthew T, Fisher Ashley A, Monks Terrence J, Lau Serrine S
机构信息
Department of Pharmacology and Toxicology, Southwest Environmental Health Sciences Center, Center for Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, USA.
出版信息
Methods Mol Biol. 2011;691:327-38. doi: 10.1007/978-1-60761-849-2_20.
The environmental toxicant hydroquinone (HQ) and its glutathione conjugates (GSHQs) cause renal cell necrosis via a combination of redox cycling and the covalent adduction of proteins within the S₃ segment of the renal proximal tubules in the outer stripe of the outer medulla (OSOM). Following administration of 2-(glutathion-S-yl)HQ (MGHQ) (400 μmol/kg, i.v., 2 h) to Long Evans (wild-type Eker) rats, Western analysis utilizing an antibody specific for quinol-thioether metabolites of HQ revealed the presence of large amounts of chemical-protein adducts in both the OSOM and urine. By aligning the Western blot film with a parallel gel stained for protein, we can isolate the adducted proteins for LC-MS/MS analysis. Subsequent database searching can identify the specific site(s) of chemical adduction within these proteins. Finally, a combination of software programs can validate the identity of the adducted peptides. The site-specific identification of covalently adducted and oxidized proteins is a prerequisite for understanding the biological significance of chemical-induced posttranslational modifications (PTMs) and their toxicological significance.
环境毒物对苯二酚（HQ）及其谷胱甘肽共轭物（GSHQs）通过氧化还原循环以及外髓质外层条纹（OSOM）中肾近端小管S₃段内蛋白质的共价加合作用，导致肾细胞坏死。给Long Evans（野生型Eker）大鼠静脉注射2-(谷胱甘肽-S-基)对苯二酚（MGHQ）（400 μmol/kg，持续2小时）后，利用针对HQ喹啉-硫醚代谢物的特异性抗体进行蛋白质印迹分析，结果显示在OSOM和尿液中均存在大量化学-蛋白质加合物。通过将蛋白质印迹膜与平行的蛋白质染色凝胶对齐，我们可以分离出加合蛋白质用于液相色谱-串联质谱（LC-MS/MS）分析。随后的数据库搜索可以确定这些蛋白质中化学加合的特定位点。最后，结合软件程序可以验证加合肽段的身份。共价加合和氧化蛋白质的位点特异性鉴定是理解化学诱导的翻译后修饰（PTMs）的生物学意义及其毒理学意义的先决条件。
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