PROJECT SUMMARY Reactive sulfur species such as persulfides (RSSH) and hydrogen polysulfides (H2Sn) play regulatory roles in redox biology. Modulation of their cellular levels could have potential therapeutic value. However, their exact mechanisms of action are still unclear. A number of fundamental questions concerning the chemistry of these species must be addressed before we can expect a biological understanding or clinical applications. It is vital to understand the reactivity of these species and to appreciate the limitation and errors that may be generated when measuring them in biological samples. In this context, research tools for accurate and convenient detection of these sulfur species are urgently needed. Our lab has recently discovered some unique chemistry and reactions of RSSH and H2Sn. Based on these discoveries, we expect that highly sensitive and specific chemical tools for the study of RSSH and H2Sn can be developed. We also expect these tools will enable us to better understand the biological significance of RSSH and H2Sn. In this application, we plan to pursue three specific aims: 1) to develop new RSSH generation methods and labeling strategies for quantitative proteomics characterization of protein S-sulfhydration, 2) to establish new chemistry and imaging probes for hydrogen polysulfides; 3) to assess the distinctive contribution of reactive sulfur species on protein S-sulfhydration and its implications on stress regulation. The proposed research will allow us to establish novel reagents and protocols for generating specific types of reactive sulfur species, new probes for imaging such species, and optimized quantitative proteomics approaches for effective profiling of protein posttranslational modifications impacted by reactive sulfur species.