Quantitative profiling of protein sulfhydrome by the BTA-TMT

The redox-based modification of cysteine residues in proteins regulates their function in many biological processes. The gas molecule H2S has been shown to sulfhydrate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent. Here we developed TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. With this approach, we revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as novel sulfhydration substrate in pancreatic beta cells. Moreover, we identified metabolic pathways selectively being targets for H2S. Using this approach, we found that the enzymes involved in cellular energy pathway are primarily subjected to a Redox Thiol Switch (RTS), which is a switch of one modification to another on the same cysteine residue. We further showed that protein S-glutathioinylation, a specific reversible thiol modification in cysteine residues under oxidative stress conditions, which can be switched to S-sulfhydration. We propose that a RTSGS from S-glutathioinylation to S-sulfhydration is a key mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.