A Fast and Ratiometric Method for Quantification of Cysteine-Bound Persulfides Based on Alkylation and Gel-Shift...

Cysteine-bound persulfides (Cys-SSH) in proteins are sulfur carrier intermediates in the synthesis of essential cofactors such as iron-sulfur clusters, molybdenum cofactor, vitamin (thiamine), and thionucleosides (thiolated tRNA). Protein-bound persulfides are also used for signaling purposes as a carrier of the "H₂S" signal. Several methods have been developed to detect and quantify cysteine-bound persulfides in protein and monitor their exchange. The main challenge in developing these techniques is to discriminate persulfidated cysteine from cysteine and other cysteine modifications. It is also critical to develop ratiometric methods to quantify the level of persulfidation in the protein of interest. We describe here a ratiometric method to label and quantify protein-bound persulfides relying on alkylation and gel-shift assays. This method is based on the derivation of cysteine and persulfides with "heavy" alkylating agents, followed by specific cleavage of the sulfur-sulfur bond of the alkylated persulfide by a reducing agent and separation of the alkylated species by electrophoresis. A persulfide is thus revealed by the appearance of a species lacking one alkylation unit under reducing conditions. We call this alkylation-reduction band-shift (ARBS) assay. Moreover, the quantification of the bands corresponding to the persulfidated and non-persulfidated species in the same lane provides a ratiometric quantification allowing determination of the level of persulfidation of individual cysteine. Other cysteine modifications such as disulfides, sulfenic, sulfinic, sulfonic acids, nitrosothiols, and sulfenamides preclude alkylation. Thus, they may appear as false positives, but they are ruled out by the analysis under nonreducing conditions since these species do not behave as persulfides under these conditions.