Proteomics Research Group, Department of Biochemistry, University College Cork, Cork, Ireland.
Talanta. 2010 Feb 15;80(4):1569-75. doi: 10.1016/j.talanta.2009.10.047. Epub 2009 Oct 31.
Protein thiols contribute significantly to antioxidant defence and selective oxidation of cysteines is important in signal transduction even in sub-stress scenarios. However, cysteine is the second rarest residue in proteins and it can be difficult to target low-abundance thiol (-SH)-containing proteins in proteomic separations. Activated thiol sepharose (ATS) allows covalent selection of -SH-containing proteins which can then be recovered by reduction with mercaptoethanol or dithiothreitol. This is a robust method for enriching -SH-containing proteins. We have used ATS to estimate the percentage (by weight) of thiol-containing proteins in cell extracts from a range of biological sources: a bacterium, Escherichia coli; a fungus, Trichoderma harzianum; and a bivalve mollusc Mytilus edulis. -SH-containing proteins account for 2.52% (E. coli), 1.4% (T. harzianum) and 1.4% (M. edulis) of total protein. Exposure to pro-oxidants did not materially alter these values. On removal of low M(r) thiols such as glutathione, the values for M. edulis did not significantly change but those for T. harzianum increased threefold. The two-dimensional electrophoresis profiles of ATS-selected proteins for each organism were compared in control and pro-oxidant-exposed preparations. This revealed that some proteins present in controls were absent in pro-oxidant-treated extracts which we attribute to thiol oxidation. ATS has significant potential in enrichment for -SH-containing proteins in redox proteomics.
蛋白质巯基对抗氧化防御有重要贡献，即使在亚应激情况下，半胱氨酸的选择性氧化在信号转导中也很重要。然而，半胱氨酸是蛋白质中第二稀有残基，在蛋白质组学分离中，靶向低丰度含巯基（-SH）的蛋白质可能很困难。活化的巯基琼脂糖（ATS）允许半胱氨酸-含有蛋白质的共价选择，然后可以通过用巯基乙醇或二硫苏糖醇还原来回收。这是一种富集含巯基蛋白质的可靠方法。我们已经使用 ATS 来估计一系列生物来源的细胞提取物中含巯基蛋白质的百分比（按重量计）：一种细菌，大肠杆菌；一种真菌，哈茨木霉；和一种双壳类软体动物贻贝。含巯基蛋白质占总蛋白质的 2.52%（大肠杆菌）、1.4%（哈茨木霉）和 1.4%（贻贝）。暴露于促氧化剂不会显著改变这些值。在去除低分子量巯基（如谷胱甘肽）后，贻贝的值没有显著变化，但哈茨木霉的值增加了三倍。每种生物的 ATS 选择的蛋白质的二维电泳图谱在对照和促氧化剂暴露的制剂中进行了比较。这表明，一些在对照中存在的蛋白质在促氧化剂处理的提取物中不存在，我们归因于巯基氧化。ATS 在氧化还原蛋白质组学中富含含巯基蛋白质方面具有重要潜力。
