Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry

Highlights

• Mass spectrometry and click chemistry can assess mitochondrial membrane potential

• This approach can be applied to investigate membrane potential in cells and in vivo

• Hypotheses dependent on small changes in membrane potential can be tested

Summary

The mitochondrial membrane potential (Δψ_m) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψ_m within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψ_m and the plasma membrane potential (Δψ_p). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by “click” chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψ_m and Δψ_p, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo.