SummaryHydrogen peroxide (H2O2) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H2O2 in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H2O2 levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H2O2 to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H2O2 that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H2O2 with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H2O2 correlates with aging, it may not be causative.

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.

Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity.

Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

A new cyclic nitrone spin trap, [4-(3′,3′-dibutyl-2′-oxy-3′H-isoindol-5′-yloxy)butyl]triphenylphosphonium bromide (MitoSpin), bearing a lipophilic cation has been prepared by a route that involves a novel Parham-type lithiation–cyclization of an isocyanate to give the isoindolinone core. MitoSpin accumulates in a membrane potential dependent way in energized mitochondria and its oxidation could potentially be used in the study of oxidative stress resulting from reactive oxygen species generated in mitochondria.

Present Investigation The object of the investigation was to compare the effect of an injection of 5 ml. 2 per cent. procaine (Injection "P") with that of a combined injection of 50 mg. hydrocortisone and 5 ml. 2 per cent. procaine (Injection "H").

Caged versions of the most common mitochondrial uncouplers (proton translocators) have been prepared that sense the reactive oxygen species (ROS) hydrogen peroxide to release the uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) from caged states with second order rate constants of 10 (±0.8) M−1 s−1 and 64.8 (±0.6) M−1 s−1, respectively. The trigger mechanism involves conversion of an arylboronate into a phenol followed by fragmentation. Hydrogen peroxide-activated uncouplers may be useful for studying the biological process of ageing.

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