biostudies-literatureMiljkovic JLMedical Research CouncilWellcome Trust85https://www.ebi.ac.uk/biostudies/studies/S-EPMC12769671biostudies-literatureUnknown17(1)H2S reversibly modifies low molecular weight (LMWSH) 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-. LMWS(S)nNature communicationsSimultaneous and sensitive quantification of protein and low molecular weight persulfides, polysulfides and H2S in biological samples.PMC12769671220257/B/20/Z221604/Z/20/ZMC_UU_00028/4110158/Z/15/Z220257/Z/20/ZMiljkovic JLBurger NKrieg THartley RCYu CSJones SAAksentijevic DCaldwell STWarrington SLee JJMurphy MPHarkiss AHJames AMfalseSimultaneous and sensitive quantification of protein and low molecular weight persulfides, polysulfides and H2S in biological samples.H2S reversibly modifies low molecular weight (LMWSH) 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-. LMWS(S)n2025-01-01T00:00:00Z2025 Dec2026-06-06T10:54:48.622Z2026-05-29T03:12:30.488ZS-EPMC127696714134536510.1038/s41467-025-66795-5