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

Medical Research Council, Mitochondrial Biology Unit, Cambridge, UK.
Nature Protocol (Impact Factor: 9.67). 05/2012; 7(5):946-58. DOI: 10.1038/nprot.2012.035
Source: PubMed


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.

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Available from: Angela Logan, Oct 24, 2014
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    • "and kept at -20 °C. Methods for extraction, quantification and analysis of the probe were adapted from [6]. Extractions were processed in 4 batches, each containing two blank control fish plus 10 experimental fish (combining 2 of the batches of five fish processed for the measurement of SMR and exposure to the probe). "
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    • "The lipophilic TPP mediates rapid uptake of MitoB into cells and accumulation in mitochondria, where MitoB reacts with H 2 O 2 and forms a phenol-derivative MitoP. MitoP/MitoB ratio can be measured by ratiometric mass spectrometry and reflects the mitochondrial H 2 O 2 level (Cochemé et al., 2012). Mutator iPSCs showed an increase in the MitoP/MitoB ratio, consistent with an increase in mitochondrial H 2 O 2 (Figure 1C). "
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    ABSTRACT: mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS)/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs) and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 05/2015; 11(10). DOI:10.1016/j.celrep.2015.05.009 · 8.36 Impact Factor
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    • "There is a wide range of compounds with different fluorophores (Dickinson et al., 2010) and adapted structures to enable targeting to mitochondria (Dickinson et al., 2013) and other compartments, such as nuclei and endoplasmic reticulum (ER) (Srikun et al., 2008; Dickinson et al., 2011; Woolley et al., 2012). In particular, combining boronate-phenol chemistry with mitochondria-targeting functional group, such as positively charged phosphonium moiety , led to generation of Mitochondrial Peroxy Yellow (MitoPY1), SHP-Mito, and MitoBoronic acid (MitoB) (Cocheme et al., 2012; Masanta et al., 2012; Dickinson et al., 2013). SHP-Mito is also a ratiometric probe and allows for increased penetration depth and prolonged imaging time using two-photon microscopy (Masanta et al., 2012). "
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