Use of dimedone-based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox-sensitive proteins.
Use of dimedone-based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox-sensitive proteins.
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DOI：
10.1016/s0076-6879(10)73003-2
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发表时间：
2010
影响因子：
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通讯作者：
Poole, Leslie B.
中科院分区：
文献类型：
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作者：
Klomsiri, Chananat;Nelson, Kimberly J.;Bechtold, Erika;Soito, Laura;Johnson, Lynnette C.;Lowther, W. Todd;Ryu, Seong-Eon;King, S. Bruce;Furdui, Cristina M.;Poole, Leslie B.
亚磺酸是半胱氨酸残基与过氧化氢反应的中间产物，在酶的催化和调节中起重要作用，也参与转录因子和其他信号蛋白的氧化还原调节。因此，在过去的几年里，人们对鉴定蛋白质磺酸的兴趣有了很大增长。二甲酮专门捕获磺酸，为合成一组新型化合物提供了基础，这些化合物将1，3-环己二酮衍生为二酮类似物，具有生物素等报告标记用于亲和力捕获，荧光标记用于视觉检测。这些试剂可以识别对氧化敏感的半胱氨酸位点和蛋白质，并可以识别发生这种氧化的细胞条件。我们已经证明了这些化合物对磺酸具有反应性和特异性，并且使用凝胶分析或质谱仪可以高灵敏度地检测到标记的蛋白质。在这里，我们进一步表征了这些试剂，表明DCP-Bio1对三种含有磺酸的蛋白质，木瓜蛋白酶，大肠杆菌fRMsr和鼠伤寒沙门氏菌过氧化还原蛋白AhpC的参入率有显著不同，并且在fRMsr的情况下，不受缓冲液pH从5.5到8.0的变化的影响。我们还提供了在细胞蛋白质中标记蛋白质磺酸的方案，既可以通过完整细胞的原位标记，也可以通过裂解时的标记。我们发现，在裂解缓冲液中加入烷基化试剂和过氧化氢酶是防止细胞裂解后形成磺酸的关键。这里提供的数据还表明，在标记过程中需要尽可能地标准化蛋白质和试剂的浓度。最后，我们介绍了几种新的测试或控制蛋白，它们可以用来评估标记过程和效率。
Sulfenic acids, formed as transient intermediates during the reaction of cysteine residues with peroxides, play significant roles in enzyme catalysis and regulation, and are also involved in the redox regulation of transcription factors and other signaling proteins. Therefore, interest in the identification of protein sulfenic acids has grown substantially in the past few years. Dimedone, which specifically traps sulfenic acids, has provided the basis for the synthesis of a novel group of compounds that derivatize 1,3-cyclohexadione, a dimedone analogue, with reporter tags such as biotin for affinity capture and fluorescent labels for visual detection. These reagents allow identification of the cysteine sites and proteins that are sensitive to oxidation and permit identification of the cellular conditions under which such oxidations occur. We have shown that these compounds are reactive and specific toward sulfenic acids and that the labeled proteins can be detected at high sensitivity using gel analysis or mass spectrometry. Here, we further characterize these reagents, showing that the DCP-Bio1 incorporation rates into three sulfenic acid containing proteins, papaya papain, Escherichia coli fRMsr, and the Salmonella typhimurium peroxiredoxin AhpC, are significantly different and, in the case of fRMsr, are unaffected by changes in buffer pH from 5.5 and 8.0. We also provide protocols to label protein sulfenic acids in cellular proteins, either by in situ labeling of intact cells or by labeling at the time of lysis. We show that the addition of alkylating reagents and catalase to the lysis buffer is critical in preventing the formation of sulfenic acid subsequent to cell lysis. Data presented herein also indicate that the need to standardize, as much as possible, the protein and reagent concentrations during labeling. Finally, we introduce several new test or control proteins that canbeusedtoevaluate labeling procedures and efficiencies.