Wei-Chieh HuangKai-Wen HsuPei-Hua PengWan-Ting ZengTing-Jia GuLi-Jie LinMin-Tsang HsiehDer-Yen LeeGEEN-DONG CHANG2024-09-112024-09-112024-08-2200032700https://www.scopus.com/record/display.uri?eid=2-s2.0-85201753141&origin=resultslisthttps://scholars.lib.ntu.edu.tw/handle/123456789/720869Protein S-sulfhydration involves the regulation of various protein functions, and resolving the S-sulfhydrated proteome (persulfidome) allows for a deeper exploration of various redox regulations. Therefore, we designed a reducible covalent capture method for isolating S-sulfhydrated proteins, which can analyze the persulfidome in biological samples and monitor specific S-sulfhydrated proteins. In this study, we applied this method to reveal the S-sulfhydration levels of proteins, including 3-phosphoglyceraldehyde dehydrogenase, NFκB/p65, and nucleolin. Furthermore, this technique can be used to enrich S-sulfhydrated peptides, aiding in the determination of protein S-sulfhydration modification sites. Finally, we observed that the S-sulfhydration and oxidation of nucleolin on the C543 residue correlate with its nuclear translocation, downstream regulation of p53, Bcl-xL, and Bcl-2 RNA levels and protein expression, as well as the protective function against oxidative stress. Therefore, this method may facilitate the understanding of the regulation of protein function by redox perturbation.enfalsejournal article10.1021/acs.analchem.4c027172-s2.0-85201753141