Measurement of Protein Persulfidation: Improved Tag-Switch Method

Abstract

Hydrogen sulfide (H₂S) is an endogenously produced signaling gasotransmitter, generated by the enzymes cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase. The involvement of H₂S in numerous physiological, as well as pathophysiological conditions, was established over the past decade. However, the exact mechanism(s) of regulation of the biological functions by H₂S are under active investigations. It is proposed that the oxidative posttranslational modification of protein cysteine residues, known as persulfidation, could be the main mechanism of action of H₂S. Protein persulfides show similar reactivity to thiols, which represents one of the main obstacles in the development of a reliable method for detection of this specific protein modification. Subsequently, having a selective method for persulfide detection is of utmost importance in order to fully understand the physiological and pathophysiological role of H₂S. Several methods have been proposed for the detection of protein persulfidation, all of which are highlighted in this chapter. Furthermore, we provide a detailed description and protocol for the first selective persulfide labeling method, a tag-switch method, developed in our group.

Keywords: CN-BOT; CN-Cy3; Gasotransmitter; Hydrogen sulfide; MSBT; Oxidative posttranslational modification; Persulfide; Tag-switch assay.