Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H 2 O 2 in living Drosophila

The role of hydrogen peroxide (H 2 O 2 ) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H 2 O 2 in vivo . Here we describe a method for assessing changes in H 2 O 2 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 H 2 O 2 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 H 2 O 2 . 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 H 2 O 2 in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H 2 O 2 production in mouse and cell culture studies.