A selective and sensitive method for quantification of endogenous polysulfide production in biological samples

Hydrogen sulfide (H₂S) is a gasotransmitter that regulates cellular homeostasis and impacts on multiple physiological and pathophysiological processes. However, it exerts many of its biological actions indirectly via the formation of H₂S-derived sulfane sulfur species/polysulfides. Because of the high reactivity of sulfur species, the detection of H₂S-derived polysulfides in biological systems is challenging and currently used methods are neither sensitive nor quantitative. Herein, we describe a LC-MS/MS-based method that makes use of Sulfane Sulfur Probe 4 to detect endogenously generated polysulfides in biological samples in a selective, sensitive and quantitative manner. The results indicate a large variability in the activity of the H₂S-generating enzymes in different murine organs, but the method described was able to detect intracellular levels of polysulfides in the nanomolar range and identify cystathionine γ-lyase as the major intracellular source of sulfane sulfur species/polysulfides in murine endothelial cells and hearts. The protocol described can be applied to a variety of biological samples for the quantification of the H₂S-derived polysulfides and has the potential to increase understanding on the control and consequences of this gaseous transmitter.