From the ‡Department of Microbial Physiology & Molecular Biology;
From the ‡Department of Microbial Physiology & Molecular Biology.
Mol Cell Proteomics. 2018 May;17(5):1035-1046. doi: 10.1074/mcp.TIR118.000671. Epub 2018 Mar 1.
The strictly anaerobic bacterium has become one of the most problematic hospital acquired pathogens and a major burden for health care systems. Although antibiotics work effectively in most infections (CDIs), their detrimental effect on the intestinal microbiome paves the way for recurrent episodes of CDI. To develop alternative, non-antibiotics-based treatment strategies, deeper knowledge on the physiology of , stress adaptation mechanisms and regulation of virulence factors is mandatory. The focus of this work was to tackle the thiol proteome of and its stress-induced alterations, because recent research has reported that the amino acid cysteine plays a central role in the metabolism of this pathogen. We have developed a novel cysteine labeling approach to determine the redox state of protein thiols on a global scale. Applicability of this technique was demonstrated by inducing disulfide stress using the chemical diamide. The method can be transferred to any kind of redox challenge and was applied in this work to assess the effect of bile acids on the thiol proteome of We present redox-quantification for more than 1,500 thiol peptides and discuss the general difficulty of redox analyses of peptides possessing more than a single cysteine residue. The presented method will be especially useful not only when determining redox status, but also for providing information on protein quantity. Additionally, our comprehensive data set reveals protein cysteine sites particularly susceptible to oxidation and builds a groundwork for redox proteomics studies in .
严格厌氧菌已成为医院获得性病原体中最具问题性的病原体之一，也是医疗保健系统的主要负担。虽然抗生素在大多数感染（CDI）中有效，但它们对肠道微生物组的有害影响为 CDI 的反复发作铺平了道路。为了开发替代的非抗生素治疗策略，必须深入了解的生理学、应激适应机制和毒力因子的调节。这项工作的重点是解决的硫醇蛋白质组及其应激诱导的改变，因为最近的研究报告表明，氨基酸半胱氨酸在该病原体的代谢中起着核心作用。我们开发了一种新的半胱氨酸标记方法来全面确定蛋白质巯基的氧化还原状态。通过使用化学二脒诱导二硫键应激来证明该技术的适用性。该方法可以转移到任何类型的氧化还原挑战中，并在这项工作中应用于评估胆汁酸对的硫醇蛋白质组的影响。我们提出了超过 1500 个巯基肽的氧化还原定量，并讨论了具有一个以上半胱氨酸残基的肽的氧化还原分析的一般困难。所提出的方法不仅在确定氧化还原状态时非常有用，而且还可以提供有关蛋白质数量的信息。此外，我们全面的数据集揭示了对氧化特别敏感的蛋白质半胱氨酸位点，并为在中进行氧化还原蛋白质组学研究奠定了基础。
