Photochemical epoxidation for the mass spectrometric elucidation of lipid structures

Cells tightly regulate lipid structures to fulfill cellular functions and to respond to external stimuli. The biochemical details of the processes that determine lipid structure alterations are often not fully understood. In this manuscript, we report the discovery of a new epoxidation strategy of unsaturated lipids, which allows the annotation of lipid head group, fatty acid composition, C=C bond position, C=C bond geometry, and sn-isomerism when derivatized lipids are analyzed with corresponding separation and mass spectrometry methods. In addition to head group, fatty acids, C=C positions that are readily available from tandem mass spectra of epoxidized deprotonated or protonated lipids, sn-isomer compositions are revealed in MSn of alkali metal adducts of derivatized lipids. Separation of epoxidation products via reversed-phase liquid chromatography (RPLC) not only distinguishes lipid C=C position and sn-isomers, remaining non-reacted unsaturated lipid C=C bonds photo-isomerize to reveal C=C E/Z configurations. To demonstrate the capabilities of the methodology, C=C positions of LPEs, LPCs, TGs, DGs, PCs, PSs, and PEs are annotated for lipid extracts in RPLC-MS2 experiments and shotgun MSn is employed to characterize 56 PC sn-isomers in HeLa and H9c2 cell lines.