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.enfalse[SDGs]SDG3[SDGs]SDG6journal article10.1021/acs.analchem.4c027172-s2.0-85201753141