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

H 2 S reversibly modifies low molecular weight (L MW SH) and protein (PrSH) thiols to form persulfides (RSS − ) and polysulfides (RS(S) n S − ) 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 H 2 S, and the terminal sulfur atom of RSS − and RS(S) n S − as diagnostic products in biological samples. In parallel, we also trap internal S atoms of RS(S) n S − , enabling quantification of H 2 S, RSS − and RS(S) n S − . L MW S(S) n S − and PrS(S) n S − are determined simultaneously in the same sample. Glutathione (GSH) is the most abundant L MW SH so we develop an orthogonal approach to quantify GSS − , enabling corroboration of L MW SS − 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.