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

Abstract

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.

Targeting Mitochondria with Small Molecules: The Preparation of MitoB and MitoP as Exomarkers of Mitochondrial Hydrogen Peroxide

“…We recently disclosed that a mitochondria---targeted arylboronate MitoB can be used to determine directly changes in the concentration of mitochondrial hydrogen peroxide in whole organisms (Figure 7c) (49,50). We have reported the use of MitoB in living flies (Drosophila melanogaster), nematodes (Caenorhabditis elegans), and mice, including providing a detailed step by step protocol (33,49,50), and are now investigating its application to fish. Since these reports include detailed descriptions of how MitoB is employed, we will only provide a very brief overview of the main features of this probe.…”

“…This was a serious short---coming as ROS are implicated in almost all the diseases of older age and the free radical theory of aging rests on the proposal that damage from mitochondrial ROS accumulates with age. We recently disclosed that a mitochondria---targeted arylboronate MitoB can be used to determine directly changes in the concentration of mitochondrial hydrogen peroxide in whole organisms (Figure 7c) (49,50). We have reported the use of MitoB in living flies (Drosophila melanogaster), nematodes (Caenorhabditis elegans), and mice, including providing a detailed step by step protocol (33,49,50), and are now investigating its application to fish.…”

Letter by Stocker and Maghzal Regarding Article, “Mitochondrial DNA Damage Can Promote Atherosclerosis Independently of Reactive Oxygen Species Through Effects on Smooth Muscle Cells and Monocytes and Correlates With Higher-Risk Plaques in Humans”

“…4 The authors interpret their data as suggestive of mitochondrial dysfunction and mitochondrial DNA defects promoting atherosclerosis independently of ROS. Similarly, the editorial 2 concludes that the concept of "mitochondrial DNA damage and oxidative stress leading to atherosclerosis may need to be revised."…”

“…Accordingly, it is suggested that MitoB be administered via intravenous injection, that the tissue of interest be collected 3 to 6 hours later, and that internal standards be added at the time of homogenization of the tissue. 4 However, none of these recommendations have been experimentally validated in mice. 1,4,5 Therefore, even when assuming that the recommended procedure was followed by Yu et al, 1 the possibility that MitoB becomes oxidized to MitoP in the circulation, that is, before it enters cells within the aortic wall, has not been excluded, just as it is unclear whether reliable and pathologically meaningful information can be obtained from a single time point measurement.…”

“…Second, experimental details concerning the use of MitoB are missing, and the methodology article cited 4 is based on studies in flies, 5 not mice. Cochemé et al 4 make recommendations for the use of MitoB in mice.…”