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

Nature Protocols volume 7, pages 946–958 (2012)Cite this article

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Abstract

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.

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Figure 1: Outline of the strategy used to assess H2O2 within living flies using MitoB.
Figure 2: Photographs of the injection procedure.
Figure 3: Time course of MitoB within flies after injection.
Figure 4: Typical calibration curves for MitoB and MitoP detection by LC-MS/MS in Drosophila.
Figure 5: Typical LC-MS/MS chromatograms for the analysis of MitoB and MitoP within flies.
Figure 6: Typical data for the analysis of MitoB, MitoP and the MitoP/MitoB ratio within flies.

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Acknowledgements

This work was supported by the UK Biotechnology and Biological Sciences Research Council, the UK Medical Research Council, the Wellcome Trust, the New Zealand Foundation for Research Science and Technology, the University of Glasgow (to C.Q.), the United Mitochondrial Disease Foundation, the Lloyds TSB Foundation for Scotland and the Royal Society of Edinburgh (to S.J.M.).

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Authors and Affiliations

  1. Medical Research Council, Mitochondrial Biology Unit, Cambridge, UK

    Helena M Cochemé, Angela Logan, Tracy A Prime, Irina Abakumova, Ian M Fearnley, Andrew M James & Michael P Murphy

  2. Department of Genetics, Evolution and Environment, Institute of Healthy Ageing, University College London, London, UK

    Helena M Cochemé, Jigna V Patel & Linda Partridge

  3. Centre for the Chemical Research of Ageing, WestCHEM School of Chemistry, University of Glasgow, Glasgow, UK

    Caroline Quin, Stephen J McQuaker & Richard C Hartley

  4. Department of Chemistry, University of Otago, Dunedin, New Zealand

    Carolyn M Porteous & Robin A J Smith

  5. Department of Biochemistry, University of Otago, Dunedin, New Zealand

    Carolyn M Porteous

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  1. Helena M Cochemé
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Contributions

H.M.C., M.P.M., R.A.J.S., L.P. and R.C.H. designed the research; H.M.C., T.A.P., A.L., I.A., C.Q., S.J.M., J.V.P. and C.M.P. carried out the experiments; H.M.C., M.P.M., A.M.J. and I.M.F. analyzed data; H.M.C., A.L. and M.P.M. wrote the paper.

Corresponding author

Correspondence to Michael P Murphy.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Video of a fly being injected with MitoB. A female fly is aneasthetised on a CO2 pad, then a glass microinjector needle containing the MitoB solution supplemented with blue dye is inserted into the thorax, using a fine-haired paintbrush to position and support the fly. The injection pulse is triggered by a foot pedal, and the blue dye rapidly diffuses throughout the fly body. (MOV 7744 kb)

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Cochemé, H., Logan, A., Prime, T. et al. Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila. Nat Protoc 7, 946–958 (2012). https://doi.org/10.1038/nprot.2012.035

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