Simultaneous and sensitive quantification of protein and low molecular weight persulfides, polysulfides and H₂S in...

H₂S reversibly modifies low molecular weight (L_MWSH) and protein (PrSH) thiols to form persulfides (RSS^-) and polysulfides (RS(S)_nS^-) for antioxidant defence and regulation of activity. However, our understanding of the biological significance of these processes is hampered by our inability to quantify these modifications. We develop a sensitive LC-MS/MS procedure that traps the sulfur atom of H₂S, and the terminal sulfur atom of RSS^- and RS(S)_nS^- as diagnostic products in biological samples. In parallel, we also trap internal S atoms of RS(S)_nS^-, enabling quantification of H₂S, RSS^- and RS(S)_nS^-. L_MWS(S)_nS^- and PrS(S)_nS^- are determined simultaneously in the same sample. Glutathione (GSH) is the most abundant L_MWSH so we develop an orthogonal approach to quantify GSS^-, enabling corroboration of L_MWSS^- measurements by sulfur atom trapping. We demonstrate in systems from proteins to ex vivo tissues how these approaches enable exploration of persulfidation in biological systems.