Assessment of H(2)S in vivo using the newly developed mitochondria-targeted mass spectrometry probe MitoA

Hydrogen sulfide (H(2)S) is produced endogenously in vivo and has multiple effects on signaling pathways and cell function. Mitochondria can be both an H(2)S source and sink, and many of the biological effects of H(2)S relate to its interactions with mitochondria. However, the significance of mitochondrial H(2)S is uncertain, in part due to the difficulty of assessing changes in its concentration in vivo. Although a number of fluorescent H(2)S probes have been developed these are best suited to cells in culture and cannot be used in vivo. To address this unmet need we have developed a mitochondria-targeted H(2)S probe, MitoA, which can be used to assess relative changes in mitochondrial H(2)S levels in vivo. MitoA comprises a lipophilic triphenylphosphonium (TPP) cation coupled to an aryl azide. The TPP cation leads to the accumulation of MitoA inside mitochondria within tissues in vivo. There, the aryl azido group reacts with H(2)S to form an aryl amine (MitoN). The extent of conversion of MitoA to MitoN thus gives an indication of the levels of mitochondrial H(2)S in vivo. Both compounds can be detected sensitively by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tissues, and quantified relative to deuterated internal standards. Here we describe the synthesis and characterization of MitoA and show that it can be used to assess changes in mitochondrial H(2)S levels in vivo. As a proof of principle we used MitoA to show that H(2)S levels increase in vivo during myocardial ischemia.