Ozonization for MALDI-2 MS imaging of carbon-carbon double bond positional isomers of phosphatidylethanolamines in...

Offline on-tissue ozonization combined with subsequent matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) offers a robust approach for spatial differentiation of carbon-carbon double bond (db) positional isomers of phospholipids across various tissue types. However, isomeric imaging of certain physiologically important lipid classes, such as phosphatidylethanolamines (PEs), remains challenging due to the comparatively low abundance of the produced ozonide ions. In this work, we therefore employed MALDI with laser post-ionization (MALDI-2) to boost PE ozonide signals and obtain new biological insights by its application on biological tissue samples.

Analysis of PE standard and PE in model tissue showed that MALDI-2 process does not significantly contribute to the in-source fragmentation of fragile ozonides produced offline unless very high post-ionization laser energies are employed. The capabilities of the developed method were demonstrated on MALDI-2 MSI mapping of positional PE isomers in whole-body sections of mouse embryos, as well as in human colorectal cancer tissue biopsies. For example, we identified three positional db isomers of PE 34:1 (Δ7, Δ9, and Δ11) and four isomers of PE 36:1 (Δ5, Δ7, Δ9, and Δ11) that exhibited distinct spatial distributions, particularly across different organs of the mouse embryo. A noteworthy observation was also the accumulation of all identified PE isomers in the brown fat tissue (BAT) regardless of the double-bond position. Within the colorectal carcinoma sample, particular enhancement of PE 36:1 (Δ9) isomer was found in the tumor tissue.

The combination of offline tissue section ozonization with MALDI-2 laser post-ionization opens a way for isomeric imaging of less abundant or poorly ionized lipids in MALDI and holds potential for elucidating the biological roles of lipid db isomers in healthy and cancer metabolism. Thanks to the offline nature of the reaction, the technique does not require any instrumental modifications of the MALDI mass spectrometer, which makes it readily adoptable.