Light-Reactive Norharmane Derivatization of Lipid Isomers by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

The lipidome, encompassing the comprehensive lipid fingerprint of a biological system, includes thousands of unique isomeric and isobaric lipid species. Mass spectrometry (MS) is an effective technique for characterizing the lipidome, although the resolution of isomeric lipid species through MS typically requires specialized or modified equipment. In this study, we introduce a novel matrix derivatization technique that leverages the unique photoreactive properties of unsaturated lipids to reveal the double-bond location in conventional matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) experiments. The principle mechanistic framework of this technique is type II photosensitization, where the MALDI matrix norharmane acts as an organic photosensitizer to generate singlet oxygen upon light exposure. The singlet oxygen then reacts with unsaturated lipid species, forming hydroperoxide derivatives at acyl group carbon double bonds, facilitating their identification. The labile nature of these hydroperoxide-functionalized lipids allows for further decomposition under normal MALDI laser exposure, enhancing the analytical resolution of isomeric lipids without additional experiments. With this approach, we were able to distinguish the 18:1 (Δ6-cis) and 18:1 (Δ9-cis) PC lipid isomers. We also demonstrated that the approach works in an imaging context, mapping lipid species in both mouse tissue and 3D cell cultures.