Novel mitochondrial and cytosolic purification pipeline for compartment-specific metabolomics in mammalian disease model tissues

Introduction Mitochondria represent an important milieu for studying the pathogenesis of several major diseases. The need for organelle-level metabolic resolution exists, as mitochondrial/cytosolic metabolites are often diluted beyond detection limits in complex samples. Compartment-specific studies are still hindered by the lack of efficient, cost-effective fractioning methods—applicable to laboratories of all financial/analytical standing. Objectives We established a novel mitochondrial/cytosolic purification pipeline for complimentary GC-TOF–MS and 1 H-NMR metabolomics using robust, commercially available fractionation strategies. Methods Magnetic based mitochondria isolation kits (MACS) were adapted for this purpose, accompanied by cytosolic filtering. Yield was assessed through the percentage recovery of citrate synthase (CS; a mitochondrial marker), purity by immunoblotting against compartment-specific proteins and integrity interrogated through the respiratory coupling ratio (RCR). The effects of the kit-based buffers on MS/NMR analyses of pure metabolite standards were evaluated. Finally, biological applicability to mammalian disease models was shown using Ndufs4 mouse brain tissue. Results With minor modifications, MACS produced around 60% more mitochondria compared to a differential centrifugation method. Less than 15% of lysosomal LAMP-2 protein was found in the MACS isolates, confirming relative purity—while RCR’s above 6 indicate sufficient mitochondrial integrity. The filtering approach effectively depleted mitochondria from the cytosolic fraction, as indicated by negligible Hsp60 and CS levels. Our GC–MS pilot yielded 60–70 features per fraction, while NMR analyses could quantify 6–10 of the most abundant compounds in each fraction. Conclusion This study provides a simple and flexible solution for mitochondrial and cytosolic metabolomics in animal model tissues, towards large-scale application of such methodologies in disease research.