Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H₂O₂ in living <i>Drosophila</i>

The role of hydrogen peroxide (H2O2) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H2O2 in vivo. Here we describe a method for assessing changes in H2O2 within the mitochondrial matrix of living Drosophila. We use a ratiometric mass spectrometry probe, MitoB ((3-hydroxybenzyl)triphenylphosphonium bromide), which contains a triphenylphosphonium cation component that drives its accumulation within mitochondria. The arylboronic moiety of MitoB reacts with H2O2 to form a phenol product, MitoP. On injection into the fly, MitoB is rapidly taken up by mitochondria and the extent of its conversion to MitoP enables the quantification of H2O2. To assess MitoB conversion to MitoP, the compounds are extracted and the MitoP/MitoB ratio is quantified by liquid chromatography–tandem mass spectrometry relative to deuterated internal standards. This method facilitates the investigation of mitochondrial H2O2 in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H2O2 production in mouse and cell culture studies