Novel Lipid Identification Using Electrospray Ionization Time of Flight Mass Spectroscopy

As part of the LIPIDMAPS consortium we have initiated a project to identify the novel lipids of the mouse macrophage RAW 264.7. To develop techniques and methods to facilitate novel lipid identification we have begun surveying the Escherichia coli and Saccharomyces cerevisiae lipidome for novel lipids. We have developed extraction and fractionation procedures and begun analyzing those lipid extracts using negative‐ion electrospray ionization time of flight mass spectroscopy. Initial hurdles to lipid identification have been the presence of solvent contaminants that hinder the identification of low abundance species or covalently modify abundant lipid species. One such artifact was structurally characterized and found to be an ethyl carbamate of phosphatidylethanolamine, formed by the reaction of the free amine of PE with phosgene and ethanol. This phosgene artifact can be avoided by substituting methylene chloride for chloroform in extraction mixtures. Use of LCMS systems further reduces the noise generated by solvent contaminants. Systematic comparison of mutant and wild‐type strains should reveal novel lipid ions, the structures of which can be identified by a combination of collision‐induced decomposition mass spectroscopy of selected ions and NMR analysis of the lipid of interest. We are poised to develop techniques to allow high throughput analyses of libraries of strains such as the E. coli Kohara overexpression library or the yeast genome ORF deletion strains. This work was supported in part by the LIPID MAPS Large Scale Collaborative Grant number GM069338 from the NIH.