A biochemical approach towards subcellular, label-free molecular imaging

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ABSTRACT Mass spectrometry-based spatial analysis has enabled label-free investigation of a broad range of endogenous biomolecules—lipids, peptides/proteins, metabolites, and glycans—in intact biological and clinical specimens. Rapid development in this area has contributed enormously to the current spatial omics research. However, the routine spatial resolution of MALDI-based mass spectrometry imaging, arguably one of the most widely used mass spectrometry imaging techniques today, is about tens of micrometers on a conventional mass spectrometer. This resolution limit imposes a roadblock towards label-free analyses of specimens at single-cell or subcellular level in existing mass spectrometry laboratories. Recent development of expansion microscopy has seen considerable success in generating fluorescence images of biological structures beyond the intrinsic spatial resolution of an optical system. Such capability has led to democratization of super-resolution fluorescence microscopy in biological and clinical laboratories. However, current expansion microscopy protocols and chemistries are not compatible with mass spectrometry imaging or analyses. The main objective of this proposal is to establish a biochemical process of sample expansion to push the spatial resolution of existing mass spectrometry imaging pipelines to the subcellular regime, namely, to a few micrometers and beyond. Towards this goal, we will develop a sample polymerization and digestion protocol optimized for mass spectrometry imaging, in which the endogenous lipids and proteins are tethered to a swelling polymer network and can be subsequently analyzed on a MALDI mass spectrometer. Our experimental approaches include (1) development of a biochemical pipeline of tissue polymerization and expansion for lipid mass spectrometry imaging; (2) extension of the pipeline to multiplexed lipid and protein detection and imaging. Successful completion of this proposal will provide the community with a label-free, subcellular-resolution molecular imaging modality without modifying the existing instrumentation.