Wojdyla Katarzyna, Williamson James, Roepstorff Peter, Rogowska-Wrzesinska Adelina
Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark.
Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark.
J Proteomics. 2015 Jan 15;113:415-34. doi: 10.1016/j.jprot.2014.10.015. Epub 2014 Oct 29.
Redox homeostasis is essential for normal function of cells and redox imbalance has been recognised as a pathogenic factor of numerous human diseases. Oxidative modifications of cysteine thiols modulate function of many proteins, mediate signalling, and fine-tune transcriptional and metabolic processes. In this study we present the SNO/SOH TMT strategy, which enables simultaneous analysis of two different types of cysteine modification: S-nitrosylation (SNO) and S-sulfenylation (SOH). The method facilitates quantitation of modification changes corrected by changes in protein abundance levels and estimation of relative modification site occupancy in a single nLC-MSMS run. The approach was evaluated in vivo using an Escherichia coli based model of mild oxidative stress. Bacteria were grown anaerobically on fumarate or nitrate. Short-term treatment with sub-millimolar levels of hydrogen peroxide was used to induce SOH. We have identified and quantified 114 SNO and SOH modified peptides. In many instances SNO and SOH occupy the same site, suggesting an association between them. High site occupancy does not equate to a site of modification which responds to redox imbalance. The SNO/SOH TMT strategy is a viable alternative to existing methods for cysteine oxidation analysis and provides new features that will facilitate our understanding of the interplay between SNO and SOH.
SNO/SOH TMT strategy outperforms other available strategies for cysteine oxidation analysis. It provides quantitative profiling of S-nitrosylation and S-sulfenylation changes simultaneously in two experimental conditions. It allows correction of modification levels by protein abundance changes and determination of relative modification site occupancy - all in a single nLC-MSMS experiment based on commercially available reagents. The method has proven precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale.
氧化还原稳态对于细胞的正常功能至关重要，氧化还原失衡已被认为是众多人类疾病的致病因素。半胱氨酸硫醇的氧化修饰可调节许多蛋白质的功能、介导信号传导并微调转录和代谢过程。在本研究中，我们提出了SNO/SOH TMT策略，该策略能够同时分析两种不同类型的半胱氨酸修饰：S-亚硝基化（SNO）和S-亚磺酰化（SOH）。该方法有助于对由蛋白质丰度水平变化校正的修饰变化进行定量，并在一次nLC-MSMS运行中估计相对修饰位点占有率。该方法在体内使用基于大肠杆菌的轻度氧化应激模型进行了评估。细菌在延胡索酸盐或硝酸盐上厌氧生长。用亚毫摩尔水平的过氧化氢进行短期处理以诱导SOH。我们已经鉴定并定量了114个SNO和SOH修饰的肽段。在许多情况下，SNO和SOH占据相同的位点，表明它们之间存在关联。高位点占有率并不等同于对氧化还原失衡作出反应的修饰位点。SNO/SOH TMT策略是现有半胱氨酸氧化分析方法的可行替代方案，并提供了新的功能，将有助于我们理解SNO和SOH之间的相互作用。
SNO/SOH TMT策略优于其他可用的半胱氨酸氧化分析策略。它在两个实验条件下同时提供S-亚硝基化和S-亚磺酰化变化的定量分析。它允许通过蛋白质丰度变化校正修饰水平并确定相对修饰位点占有率——所有这些都在基于市售试剂的一次nLC-MSMS实验中完成。该方法已被证明足够精确和灵敏，能够在蛋白质组范围内检测和定量内源性氧化应激水平。
