In situ identification of S-sulfenylation sites in Arabidopsis thaliana using the genetic YAP1 probe

Hydrogen peroxide (H2O2) reacts, directly or indirectly, with cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. This cysteine oxidation steers diverse cellular processes by altering protein interactions, trafficking, conformation, and function. We present a method to identify S-sulfenylated cysteines sites in proteins using a genetic probe based on the yeast AP-1–like (YAP1) transcription factor that specifically reacts with sulfenic acid sites to form mixed disulfides. After a tryptic digest and with the help of an antibody directed against a 7-amino-acid YAP1C-derived peptide that contains the redox-active cysteine, we enriched for disulfide-linked peptides. The mass spectral characteristics for fragment ions of the mixed disulfides made it possible to identify 1,747 S-sulfenylation protein sites in Arabidopsis under H2O2 stress. Furthermore, cross-study comparison shows that 55% of cross-linked sites match with previously reported S-sulfenylated, S-nitrosylated and reversibly oxidized cysteines in Arabidopsis. These include well-characterized redox-sensitive cysteines, such as Cys20 of DEHYDROASCORBATE REDUCTASE 2 (DHAR2) and Cys181 of MAP KINASE 4 (MAPK4). Altogether, we describe a novel approach to identify S-sulfenylated sites in situ using the YAP1C probe, thereby offering a non-invasive manner to study site-specific cysteine oxidation and which can be applied for identification of S-sulfenylated sites at the organellar level.