Making OzID go FFASTer: Combining stable-isotope tagging with ozone-induced dissociation to uncover changes in fatty acid unsaturation within neurosecretory cells

Abstract

Despite lipids providing a major contribution to the mass of the brain, at the molecular level, the brain lipidome remains incompletely described and the functions of many of the species identified to date are unknown. Recently, unusual unsaturated fatty acids –with carbon-carbon double bonds in positions not predicted by canonical lipid metabolism– were identified in the mouse brain. The extent to which these unusual lipids contribute to neurochemical processes presents a significant gap in knowledge that is challenging to close due to technological impediments to the detection and tracing of such novel fatty acids. Here we deploy state-of-the-art mass spectrometric methods coupled with diagnostic ion-molecule reactions in the form of ozone-induced dissociation to enable detection and relative quantification of unsaturated fatty acids in an in vitro brain model based on the PC12 cell line. This approach revealed the presence of abundant populations of non-canonical fatty acids, including FA 16:1n-10,cis (sapienic acid) and FA 18:1n-10,cis (dihomosapienic acid), in extracts from PC-12 cell lines. Neither of these fatty acids have previously been reported in neurons or neurosecretory-like cells, with both demonstrated to increase between 2 and 4-fold in relative abundance upon active stimulation.