The number of spatial omics technologies being developed is increasing1. However, a missing tool is one that can locate proteins in tissues in an untargeted manner at high spatial resolution and coverage. Here we present in situ imaging proteomics via expansion (iPEX), which integrates isotropic tissue magnification2 with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. iPEX provides scalable spatial resolution down to the micrometre scale and substantially increases the sensitivity of protein identification by 10–100-fold. Using the retina as a model, iPEX enabled the construction of spatial proteomic maps with high precision, the visualization of single-cell layers and extrasomatic structures and the identification of colocalized proteins. iPEX was readily applied to diverse tissues, including brain, intestine, liver and organoids, detecting 600–1,500 proteins at 1–5-µm effective pixel size. The application of iPEX to depict spatial proteomic maps in brains of mice with 5xFAD Alzheimer’s disease revealed an early-onset mitochondrial aberrancy. Notably, in young mice, the peroxisomal acetyl-CoA acyltransferase ACAA1A—of which the N392S mutant is a monogenic risk factor in Alzheimer’s disease3—was downregulated. ACAA1 depletion blocked the biosynthesis of long-chain polyunsaturated fatty acids, including docosahexaenoic acid, in multiple cellular contexts. These lipidome alterations were restored in cells overexpressing wild-type ACAA1 but not ACAA1(N392S), which suggests that the dysregulation of long-chain polyunsaturated fatty acids has an early role in neurodegeneration. Together, these results demonstrate that iPEX facilitates untargeted spatial proteomics at micrometre resolution for diverse applications.
