Fatty acid metabolome extraction from mycobacterial cells for GC-MS metabolomics analysis

Abstract

Metabolomics is becoming an increasingly popular research tool for identifying new biomarkers, which can, among other applications, be applied to elucidate various microbial growth and virulence mechanisms. Since the lipid composition of numerous microorganisms are unique and characteristic of the particular species, and in many instances also associated with several of their growth and virulence features, we developed a method for extracting the total free fatty acid metabolome from mycobacterial cells, in order to better characterize these using a gas chromatography–mass spectrometry (GC-MS) metabolomics approach. The described method can be considered an optimized Bligh–Dyer approach, since it uses the traditional solvents; chloroform, methanol and water, in a ratio of 1:2:1. However, due to the robust cell walls associated with mycobacteria, and many other microorganisms, the method was adapted to include a step which allows for the physical disruption of the cells using a vibration mill, which dramatically increases the efficiency of this approach. Hereafter, the organic phase is collected, dried, and methylated (as a derivatization step), prior to GC-MS analyses