Rapid Immunopurification Technique Enables Accurate Determination of Mitochondrial Metabolites

A rapid immunopurification technique was used to isolate mitochondria with metabolite contents that were relatively undamaged by enzyme activity and free from components of other cellular organelles.

While metabolite profiling by mass spectrometry has been widely applied for research purposes at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. However, in a recent development in this field, investigators at the Whitehead Institute (Cambridge, MA, USA) have developed a method for the rapid and specific isolation of mitochondria that allows accurate quantification of this organelle's metabolite content.

Up to now, various methods of differential centrifugation have been used to isolate mitochondria. Unfortunately, these protocols also precipitate non-mitochondrial material and other organelles. In addition, these relatively lengthy procedures promote enzymatic modifications of mitochondrial metabolites that lead to variable results.

The investigators described a novel immunopurification technique for isolation of mitochondria in the August 25, 2016, issue of the journal Cell. The method was based on antibody-covered beads that bound to specific mitochondrial surface markers. Recovery of antibody-bead-mitochondria complexes permitted rapid disruption of the mitochondria and inhibition of all enzymatic activity within 10 minutes. The immunopurification technique was used it in tandem with a database of predicted mitochondrial metabolites ("MITObolome") to measure the matrix concentrations of more than 100 metabolites across various states of mitochondrial respiratory chain (RC) function.

"The advantage of this new method is that it offers a combination of both increased speed and specificity," said senior author Dr. David Sabatini, professor of biology at the Whitehead Institute. "We are quite excited about applying this workflow in vivo and to other organelles such as lysosomes."

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