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. 2024 Dec 4;68(4):467–478. doi: 10.1042/EBC20230095

Figure 5. Schematic overview of tag-switch based persulfidation-labelling technologies.

Figure 5

(A) Modified tag-switch assay overview [67]. Free thiols and persulfides were labelled with MSBT to generate distinct products, thioesters and disulfides that possessed more enhanced electrophilicity than original disulfides. The employment of fluorescent attached-nucleophiles enabled the detection of persulfidation at specific channel without usage of reducing reagents. (B) Dimedone-switch assay proposed by Zivanovic et al. [66] NBF-Cl served to block not only free thiols and persulfides but also sulfenic acids and amino groups. Excessive usage of NBF-Cl ideally labelled all amino groups, serving as total protein amount. The addition of dimedone-based probes, DCP-Bio1 and DAz2/Cy5, allowed selective switch of disulfides formed at the persulfidation sites, for further MS/MS analysis or in-gel detection. DAz2/Cy5, Cy5 conjugated dimedone via CuAAC reaction; DCP-Bio1, biotinylated dimedone; MSBT, Methylsulfonyl benzothiazole; NBF-Cl, 4-chloro-7-nitrobenzofurazan. (Created with BioRender.com.)