Proteomics Research Group, Department of Biochemistry, Environmental Research Institute, University College Cork, Cork, Ireland.
Mar Environ Res. 2010;69 Suppl:S25-7. doi: 10.1016/j.marenvres.2009.11.005. Epub 2009 Nov 14.
If reactive oxygen species (ROS) levels exceed antioxidant defences, oxidative stress occurs; a common response to environmental pollutants. Proteins absorb ∼70% of ROS, altering amino acid side-chains. Cys (-SH) oxidises to sulphenic (-SOH), sulphinic (-SO(2)H), cysteic (-SO(3)H) acids and disulphide bridges (-S-S-). Two-dimensional electrophoresis (2DE) under-selects certain protein categories (e.g. extreme pI, small proteins) so activated thiol sepharose (ATS) was used to select sub-proteomes of thiol-containing proteins in menadione-exposed Escherichia coli. ATS bound thiol-containing proteins (but not oxidised thiols) via mixed disulphides. Tryptic digestion of bead-bound proteins was followed by LC-tandem MS. Many proteins were identified in controls with significantly fewer in menadione-treated cells (e.g. chaperonins, transcription/translation-related and ribosomal proteins; aminoacyl tRNA synthetases and metabolic enzymes. Non-denaturing ATS capture (followed by reduction) demonstrated lower specific activities of key enzymes which is attributed to thiol oxidation. This method may be generally useful in ecotoxicology for identification of oxidative stress targets.
如果活性氧（ROS）水平超过抗氧化防御能力，就会发生氧化应激；这是对环境污染物的常见反应。蛋白质吸收约 70%的 ROS，改变氨基酸侧链。半胱氨酸（-SH）氧化为亚磺酸（-SOH）、亚硫酸（-SO2H）、胱氨酸（-SO3H）酸和二硫键（-S-S-）。二维电泳（2DE）对某些蛋白质类别（如极端 pI、小蛋白质）进行了选择，因此在 menadione 暴露的大肠杆菌中使用激活的巯基琼脂糖（ATS）来选择含巯基蛋白质的亚蛋白质组。ATS 通过混合二硫键与含巯基的蛋白质（而不是氧化的巯基）结合。珠上结合的蛋白质经过胰蛋白酶消化，然后进行 LC-串联 MS。在对照中鉴定出许多蛋白质，而在 menadione 处理的细胞中显著减少（例如伴侣蛋白、转录/翻译相关和核糖体蛋白；氨酰 tRNA 合成酶和代谢酶。非变性 ATS 捕获（随后还原）表明关键酶的特异性活性降低，这归因于巯基氧化。这种方法在生态毒理学中可能普遍用于鉴定氧化应激靶标。
