Absolute Quantification of Matrix Metabolites Reveals the Dynamics of Mitochondrial Metabolism

Abstract

SUMMARY Mitochondria house metabolic pathways that impact most aspects of cellular physiology. While metabolite profiling by mass spectrometry is widely applied at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. We describe a method for the rapid and specific isolation of mitochondria, which we use in tandem with a database of predicted mitochondrial metabolites (“MITObolome”) to measure the matrix concentrations of greater than 100 metabolites across various states of respiratory chain (RC) function. Disruption of the RC revealed extensive compartmentalization of mitochondrial metabolism and signatures unique to the inhibition of each RC complex. Pyruvate enables the proliferation of RC-deficient cells, but had surprisingly limited effects on matrix contents. Interestingly, despite failing to restore matrix NADH/NAD balance, pyruvate did increase aspartate, likely through the exchange of matrix glutamate for cytosolic aspartate. We demonstrate the value of mitochondrial metabolite profiling and describe a strategy applicable to other organelles.

Metabolic control by sirtuins and other enzymes that sense NAD+, NADH, or their ratio

“…During the purification procedure, alterations in NAD + and NADH concentrations are expected. This limitation was recently overcome, at least in part, by leveraging affinity-tag-based purification of mitochondrial organelles, prior to metabolite extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [20]. In line with earlier findings, this study reported a total mitochondrial NAD + concentration of 818 μM and an NAD + /NADH ratio of 111.…”

De novo acetate production is coupled to central carbon metabolism in mammals

“…Possibly, other thiol-containing molecules, such as GSH could serve as the acetyl group acceptor, considering that the cellular CoA is around 10 µM while GSH is 1 to 7 mM (Fig. 3b), and similar concentration values in different types of cells or tissues were also reported by others [28][29][30] . Low levels of CoA and high levels of GSH were also observed in autochthonous mouse tumors providing a physiological context for this occurrence (Fig.…”

Metabolite Measurement: Pitfalls to Avoid and Practices to Follow

“…Isolation of subcellular organelles can unfortunately perturb metabolite concentrations: There is no validated method to quench metabolic activity without causing leakage between compartments. Faster organelle isolation can help mitigate such problems, but the current standard for fast isolation of ~10 min remains slow relative to turnover of many metabolites (141–143). Nevertheless, some biological insights may be obtained, although it is important to validate their accuracy.…”