High-Resolution Spatial Proteomics Uncovers Molecular Diversity across Mouse Hippocampal Subregions

The hippocampus (HP) is a structurally and functionally heterogeneous brain region critical for learning, memory, and spatial navigation. Although transcriptomic studies have revealed subregion-specific gene expression patterns, the corresponding high-resolution spatial proteomic landscape remains largely unexplored. Here, we applied a single-voxel proteomics workflow at 50 μm resolution to profile the protein composition of HP subregions, including the dentate gyrus (DG) and Cornu Ammonis fields 1–3 (CA1–CA3). Across the four subregions, we identified over 2,200 protein groups and found both conserved and region-specific proteins. Differential expression and network analyses revealed molecular signatures associated with specific subregions, including synaptic plasticity in CA1, mitochondrial metabolism in CA2, cytoskeletal organization and protein homeostasis in CA3, and excitatory signaling in DG. Integration with transcriptomic data confirmed consistent region-enriched markers at both the gene and protein levels, including Homer1 and Itpka in CA1, Homer3 in CA3, and Calb1/2 in DG. Thus, our data set demonstrates subregion-specific molecular specialization at the proteome level and provides a high-resolution resource to link structural, functional, and metabolic diversity in the hippocampus. Further application of our high-resolution spatial proteomics will lay the foundation for studying HP development, circuit function, and disease-associated proteomic alterations.