Improvements to defi ne mitochondrial metabolomics using nonaqueous fractionation

Abstract

Defi ning metabolite abundance and resulting fractional isotope enrichments, within and between cellular compartments, still remain a major challenge in modern plant biochemistry. Optimized protocols for rapid isolation of mitochondria (e.g., silicone oil centrifugation or membrane fi lters) or visualization of metabolites/ metabolic states (e.g., fl uorescence resonance energy transfer (FRET) or redox-sensitive fl uorescent markers (ro GFP)) have signifi cantly improved and expanded our knowledge regarding mitochondrial metabolism. However, the application of nonaqueous fractionation (NAQF) to separate and quantify metabolites across subcellular compartments remains popular as a nontargeted, validated approach towards studying metabolism, and provides a top-down overview of metabolite distribution across the majority of the subcellular compartments in a single preparation. Unfortunately, of all the organelles resolved using this method, the mitochondrion still remains the most poorly defi ned. Here, the development and suggested improvements to resolve the mitochondrial metabolome are described.