Deciphering the three-dimensional biomolecular distribution in the Alzheimer's disease brain: A multiomic approach integrating immunohistochemistry with MALDI MS imaging

Alzheimer's disease (AD) is characterized by complex and distinct spatial and molecular changes in the brain that contribute to its onset and progression, which includes disruptions in proteins, lipids, and neuroinflammatory pathways. In this study, we introduce a three-dimensional (3D) spatial multi-omics imaging approach that integrates lipid mass spectrometry imaging (MSI) with targeted proteomic analysis using 10-plex photo-cleavable mass tag (PC-MT) probes. Brain tissues from wild-type (WT) and AD model mice were sectioned at four coronal levels, spaced 1500 μm apart, enabling the reconstruction of a depth-resolved molecular atlas. Lipid MSI revealed region- and depth-specific alterations in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and other lipid classes between AD and WT brains. In parallel, protein MSI using PC-MT probes identified spatially distinct changes in neuroinflammatory and neurodegenerative markers across the cortex, hippocampus, and thalamus at different depths. This integrative platform uncovered co-localization patterns of lipid dysregulation and protein overexpression unique to AD pathology, highlighting vulnerable brain subregions. Our findings demonstrate that combining targeted protein and untargeted lipid imaging in 3D enables the discovery of spatially coordinated molecular signatures in neurodegeneration, offering a powerful framework for biomarker discovery and therapeutic development.