半胱氨酸反应性分析揭示植物病原体中的氧化还原调控。

Cysteine Reactivity Profiling to Unveil Redox Regulation in Phytopathogens.

机构信息

The Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford, UK.

Advanced Proteomics Centre, Department of Biochemistry, University of Oxford, Oxford, UK.

出版信息

Methods Mol Biol. 2022;2447:105-117. doi: 10.1007/978-1-0716-2079-3_9.

DOI:10.1007/978-1-0716-2079-3_9

PMID:35583776

Abstract

Reactivity-based chemical proteomics is a powerful technology based on the use of tagged chemicals that covalently react with surface-exposed residues on proteins in native proteomes. Reactivity profiling involves the purification, identification, and quantification of labeled peptides by LC-MS/MS. Here, we have detailed a protocol for reactivity profiling of Cys residues using iodoacetamide probes, displaying >1000 reactive Cys residues in the proteome of phytopathogen Pseudomonas syringae pv. tomato DC3000 (PtoDC3000). Comparative reactivity profiling of PtoDC3000 treated with or without hydrogen peroxide (HO) identified ~200 HO-sensitive Cys residues in antioxidant enzymes, metabolic enzymes, and transcription regulators. Interestingly, half of these HO-sensitive Cys residues are more reactive in response to HO and several proteins have multiple Cys residues with opposite reactivities in response to HO exposure.

摘要

基于反应性的化学蛋白质组学是一种强大的技术，它基于使用标记化学物质，这些化学物质与天然蛋白质组中表面暴露的残基发生共价反应。反应性分析包括通过 LC-MS/MS 对标记肽进行纯化、鉴定和定量。在这里，我们详细描述了使用碘乙酰胺探针进行半胱氨酸残基反应性分析的方案，在植物病原菌丁香假单胞菌 pv.番茄 DC3000（PtoDC3000）的蛋白质组中显示了 >1000 个反应性半胱氨酸残基。对用或不用过氧化氢（HO）处理的 PtoDC3000 进行比较反应性分析，在抗氧化酶、代谢酶和转录调节剂中鉴定出约 200 个对 HO 敏感的半胱氨酸残基。有趣的是，这些对 HO 敏感的半胱氨酸残基中有一半对半胱氨酸的反应性更强，并且一些蛋白质具有多个半胱氨酸残基，它们对半胱氨酸的反应性在暴露于 HO 时是相反的。