Spatial Multiomics Combining Lipids and Gene Expression Using MALDI ISH MSI

Current spatial gene expression techniques rely upon DNA microarrays, next-generation sequencing, and fluorescence microscopy, which are often costly, time-consuming, and/or restricted in the sampling area. Matrix-assisted laser desorption/ionization <i>in situ</i> hybridization (MALDI ISH) mass spectrometry imaging (MSI) combines the spatiotemporal capabilities of <i>in situ</i> hybridization with MSI using photocleavable peptide mass tags and nucleic acid probes to enable serial detection of lipidomic and gene expression data. We synthesized a copper-catalyzed azide-alkyne cycloaddition (CuAAC) <i>o</i>-nitrobenzylic azide linker that is compatible with solid phase peptide synthesis (SPPS) to enable a flexible and modular conjugation platform between DNA sequences and peptide mass tags. After conjugation of the mass tag and hybridization to the native RNA target, the triazole-functionalized <i>o</i>-nitrobenzyl linker can undergo photolytic cleavage to separate the hybridized nucleic acid sequence from its corresponding peptide mass tag. Using this approach, we synthesized 33 unique photocleavable mRNA probes and successfully validated 12 distinct gene expression patterns across sagittal sections of fresh-frozen murine brain of both wild-type and a Canavan's disease model. This method enables codetection of native RNA and lipidomic signals from the same tissue section, providing an alternative strategy for spatial multiomic analysis. While this study demonstrates a 12-plex implementation, the platform is scalable and may support expanded multiplexing with continued optimization of peptide design and instrumentation. This approach is broadly applicable across disease models and offers new insights into transcriptome-metabolome interactions.