Determining the metabolic profiles in drosophila melanogaster: development and application of a novel ion-pairing liquid chromatography-mass spectrometry protocol

Abstract

Genetic perturbations and foreign chemicals can result in a multitude of changes across a wide range of biochemical processes in a biological system. These perturbations may affect the metabolome, the small molecule metabolites in an organism. Recently, liquid-chromatography coupled to mass spectrometry (LC-MS) technology has been used to quantify large proportions of the metabolome, however standardized protocols are not yet available for use with Drosophila melanogaster. Here, I developed an ion-pairing LC-MS protocol for the metabolomic characterization of D. melanogaster and demonstrated its implementation in establishing the metabolomic profile of flies under oxidative stress and in the metabolic profiles of four different Drosophila species. I demonstrated that this new method allows for the detection of otherwise difficult metabolites and that it is repeatable and sensitive with acceptable levels of ionsuppression, matrix effects, limits of detection and quantification. I then used this method to determine and quantify the metabolomic fingerprints of loss of Superoxide dismutase activity and paraquat-induced stress. Comparing and contrasting the effects of these two sources of oxidative stress, I document both similarities and stressor-specific effects.