Detection of protein persulfidation in plants by the dimedone switch method

Abstract

Hydrogen sulfide (H₂S) 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, H₂S 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.

Keywords: Hydrogen sulfide signaling; In-gel persulfidation detection; Persulfidation; Plant signaling; Proteomics.