Mapping Double-Bond Positional Isomers of Phosphatidylcholines in Zebrafish Using OzMALDI Mass Spectrometry Imaging

Precise characterization of lipid structures requires resolving carbon-carbon double-bond (db) positional isomers, structural details that are typically inaccessible using conventional tandem mass spectrometry. To overcome this limitation, we previously developed an in-source ozonolysis method for matrix-assisted laser desorption/ionization (MALDI) by introducing ozone gas into the MALDI source. This technique, termed OzMALDI, enables confident assignment of db positions during mass spectrometry imaging (MSI). In this study, we applied the OzMALDI workflow to zebrafish to elucidate the phosphatidylcholine (PC) structures in MSI. This was achieved by combining MALDI-MS/MS of lithium adducts for fatty acyl composition analysis with OzMALDI-MS/MS for db position analysis. Using this integrated approach, we identified the majority of PC species in zebrafish for their fatty acyl chain and positional isomers, along with their localization. Monounsaturated PCs exhibited distinct n-7 and n-9 isomers with pronounced organ-specific enrichment: n-7 isomers were predominant in the heart and dorsal muscle, whereas n-9 isomers were enriched in the ovary. In contrast, di- and polyunsaturated PC isomers did not display organ-specific differences. Together, these results establish a foundation for a future investigation into lipid remodeling in wild-type zebrafish as well as generic models and disease states.