Institute of Plant Biochemistry and Photosynthesis, CSIC-University of Seville, Seville, Spain.
Leibniz Institute for Analytical Sciences, ISAS e.V., Dortmund, Germany.
Methods Enzymol. 2022;676:385-402. doi: 10.1016/bs.mie.2022.07.024. Epub 2022 Aug 18.
Hydrogen sulfide (HS) is a well-known signaling molecule in both animals and plants, endogenously produced by cells, and involved in a wide variety of biological functions. In plants, HS regulates a wide range of essential aspects of plant life, including plant responses to numerous stresses and physiological processes as important as abscisic acid (ABA)-dependent stomatal movement, photosynthesis, and autophagy. The best studied molecular mechanism responsible of sulfide signaling is protein persulfidation, a post-translational modification of cysteine residues, where a thiol group (P-SH) is transformed into a persulfide group (P-SSH). In this way, persulfidation has emerged as a new type of cellular redox mechanism that can regulate protein structure and function and interest in this modification has increased exponentially. However, the identification and the development of detection methods have been challenging. Nevertheless, on the basis of the chemical differences between the thiol and the persulfide groups, different methods have been implemented. In plants, different high-throughput proteomic analyzes have been performed using a tag-switch method where in the first step all thiols and persulfides are blocked and then in the second step persulfides are selectively labeled using a specific nucleophile. This chapter outlines a new method, previously described in mammals, that has been applied to detect persulfidation in plants and is based on the same chemical premise but consists of chemoselective persulfide labeling with dimedone-based probes. Here, we provide a detailed workflow of this method that includes procedures for the determination of the persulfidation level of a protein extract visualized and quantified by fluorescence on the gel on one side, and on the other, the labeling and purification of persulfidated proteins for identification by mass spectrometry.
硫化氢（HS）是动物和植物中一种众所周知的信号分子，由细胞内源性产生，参与广泛的生物学功能。在植物中，HS 调节植物生命的广泛的基本方面，包括植物对多种胁迫的反应和重要的生理过程，如依赖脱落酸（ABA）的气孔运动、光合作用和自噬。负责硫化物信号的最佳研究分子机制是蛋白质过硫化，这是半胱氨酸残基的一种翻译后修饰，其中一个硫醇基团（P-SH）转化为过硫化物基团（P-SSH）。以这种方式，过硫化已成为一种新的细胞氧化还原机制，可以调节蛋白质的结构和功能，对这种修饰的兴趣呈指数级增长。然而，鉴定和开发检测方法一直具有挑战性。然而，基于硫醇和过硫化物基团之间的化学差异，已经实施了不同的方法。在植物中，使用标签转换方法进行了不同的高通量蛋白质组学分析，在该方法的第一步中，所有的硫醇和过硫化物都被阻断，然后在第二步中，使用特定的亲核试剂选择性地标记过硫化物。本章概述了一种新方法，该方法以前在哺乳动物中描述过，并已应用于检测植物中的过硫化物，该方法基于相同的化学前提，但由基于二甲基酮的探针进行化学选择性的过硫化物标记。在这里，我们提供了该方法的详细工作流程，其中包括从凝胶上的荧光可视化和定量一侧确定蛋白质提取物的过硫化水平的程序，以及对过硫化蛋白质进行标记和纯化以通过质谱进行鉴定的程序。
