Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

The overall goal of the following experiment is to obtain enriched mitochondrial fractions that yield enough mitochondrial metabolites for metabolomic studies using drosophila melanogaster. This is achieved by first raising a sufficient amount of flies to generate enough metabolites for mitochondrial enriched fractions. As a second step, flies are homogenized into a glass Teflon downs homogenizer, which breaks down the cellular membrane without damaging mitochondrial integrity.

Next homogenates are subjected to differential centrifugation in order to generate enriched mitochondrial samples. The results show that pronounced mitochondrial metabolic changes were detected using this protocol, which were undetected using wholely based metabolomics analysis on mitochondrial enriched fractions and whole animal extracts. The main advantage of this procedure versus a whole fly metabolomic analysis is that isolation of mitochondria prior to metabolomic analysis allows for a increased sensitivity and detection of mitochondrial metabolite shift.