Metal Oxide Laser Ionization Mass Spectrometry Imaging (MOLI MSI) Using Cerium(IV) Oxide

Abstract

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a powerful technique for spatially resolved metabolomics. A variation on MALDI, termed metal oxide laser ionization (MOLI), capitalizes on the unique property of cerium(IV) oxide (CeO₂) to induce laser-catalyzed fatty acyl cleavage from lipids and has been utilized for bacterial identification. In this study, we present the development and utilization of CeO₂ as an MSI catalyst. The method was developed using a MALDI TOF instrument in negative ion mode, equipped with a high frequency laser. Instrument parameters for MOLI MS fatty acid catalysis with CeO₂ were optimized with phospholipid standards and fatty acid catalysis was confirmed using lipid extracts from reference bacterial strains, and sample preparation was optimized using mouse brain tissue. MOLI MSI was applied to the imaging of normal mouse brain revealing differentiable fatty acyl pools in myelinated and nonmyelinated regions. Similarly, MOLI MSI showed distinct fatty acyl composition in tumor regions of a patient derived xenograft mouse model of glioblastoma. To assess the potential of MOLI MSI to detect pathogens directly from tissue, a pseudoinfection model was prepared by spotting Escherichia coli lipid extracts on mouse brain tissue sections and imaged by MOLI MSI. The spotted regions were molecularly resolved from the supporting mouse brain tissue by the diagnostic odd-chained fatty acids and reflected control bacterial MOLI MS signatures. We describe MOLI MSI for the first time and highlight its potential for spatially resolved fatty acyl analysis, characterization of fatty acyl composition in tumors, and its potential for pathogen detection directly from tissue.

Figure 1.

Optimization of MOLI MS using the rapifleX platform. (A) Mass spectra of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) standard using CeO₂, confirming anticipated fatty acyl catalysis. (B) Signal intensity of fragmented fatty acyl chains using various concentrations of POPC and CeO₂. Left panel shows signal intensity of m/z 255 (FA 16:0, palmitic acid); right panel shows signal intensity of m/z 281 (FA 18:1, oleic acid). (C) MOLI MS of extracted lipids from reference bacterial species (and strain numbers), containing CeO₂-catalyzed fatty acyl fragments.

Figure 2.

MOLI MSI image of FA 20:1 using different applications of CeO₂.

Figure 3.

MOLI MSI of control mouse brain tissue. Coronal sections (12 µm thickness) of frozen mouse brains were thaw mounted and spotted with a solution of CeO₂ in isopropanol. MOLI-generated fatty acyl pool profiles contained in various neuroanatomical regions are highlighted. MOLI MSI images were acquired using 100 µm pixel resolution.

Figure 4.

MOLI MSI of mouse brain tissue containing tumor of glioblastoma patient-derived xenograft. Histopathology (H&E stain of tissue): tumor delineated as T, Corpus callosum as CC. MOLI MSI ions images were acquired using 100 µm pixel resolution.

Figure 5.

MOLI MSI of a coronal normal mouse brain cryosection spotted with E. coli lipid extract. CeO₂-catalyzed fatty acyl spectra demonstrating localization of bacterial-enriched acyl groups (red) in spotted hemisphere, compared with normal mouse brain spectra (blue). MOLI MSI images were acquired using 100 µm pixel resolution.