DYn-2 Based Identification of Arabidopsis Sulfenomes*

Abstract

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H 2 O 2 ) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H 2 O 2 dose-and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H 2 O 2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative posttranslational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the

Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles

“…8,12,35–38 Moreover, due to the lack of scaffold diversity among present Cys-SOH probes, current proteomics-based approaches (such as FBLD and ABPP) to identify small-molecule ligands targeting sulfenylated proteins could not be implemented effectively. 13,36,39–41 To remedy the moderate reactivity of current nucleophile probes and to increase the structure diversity, we recently developed a library of cyclic and linear C-nucleophiles that showed diverse reactivity profiles toward Cys-SOH in a small molecule dipeptide model as well as in a protein model. 42–44 The next logical step is to identify the biological target preferences of these newly developed nucleophiles.…”

The Expanding Landscape of the Thiol Redox Proteome

“…4C). This strategy identified ϳ200 potential targets of protein S-sulfenylation in the unstimulated HeLa cells or H 2 O 2 -treated Arabidopsis cells, respectively (81,82). However, these protein-level analyses did not identify specific sites of this modification, which increases the possibility of false-positive identifications.…”

What is the role of hydrogen peroxide in plant peroxisomes?

“…Additionally, xanthine oxidoreductase activity, which generates superoxide radicals, in its oxidase form (XOD) has been shown to be present in isolated pea leaf peroxisomes using electron microscopy immunogold labelling (Corpas et al 2008); however, its peroxisomal localisation is considered to be controversial by some authors as it has not yet been found in Arabidopsis peroxisomes. In this context, it must be pointed out that a combination of biochemical, cellular and proteomic approaches has significantly increased the number of new and sometimes unpredicted peroxisomal proteins (Corpas et al 1993a(Corpas et al , 2008Reumann et al 2007a,b;Akter et al 2015). More recently, a new type of bioinformatic analysis using algorithms to predict peroxisomal targeting signals (PTSs) combined with experimental validation has become a powerful tool to identify new peroxisomal protein candidates (Chowdhary et al 2012;Reumann et al 2012;Skoulding et al 2015).…”