Hydrogen sulfide-mediated signaling pathways regulates many physiological and pathophysiological processes in mammalian and plant systems. The molecular mechanism by which hydrogen sulfide exerts its action involves the post-translational modification of cysteine residues to form a persulfidated thiol motif, named as protein S-sulfhydration or persulfidation. We have developed a comparative and quantitative proteomic analysis approach for the detection of endogenous S-sulfhydrated proteins in wild-type Arabidopsis and L-CYSTEINE DESULFHYDRASE 1 mutant leaves by using the tag-switch method. Bioinformatic analysis of the identified proteins revealed that S-sulfhydrated cysteines are part of a wide range of biological functions regulating important processes such as carbon metabolism, plant responses to abiotic and biotic stresses, plant growth and development, and RNA translation. Quantitative analysis in both genetic backgrounds reveals that protein sulfhydration is mainly involved in primary metabolic pathways such as tricarboxylic acid cycle, glycolysis or Calvin cycle, suggesting that this protein modification is a new regulatory component in these pathways