Comprehensive lipid structure annotation via photochemical epoxidation and mass spectrometry

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 present 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. Tandem mass spectra of epoxidized deprotonated or protonated lipids provide information on head groups, fatty acids, and C = C positions, while MSⁿ of alkali metal adducts reveals sn-isomer compositions. 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 bovine heart and liver extracts in RPLC-MS² experiments, and shotgun MSⁿ is employed to characterize 56 PC sn-isomers in HeLa and H9c2 cell lines.