High‐Throughput Proteoform Imaging for Revealing Spatial‐Resolved Changes in Brain Tissues Associated with Alzheimer's Disease

Abstract Spatially resolved characterization of proteoforms has substantial potential to significantly advance the understanding of physiological and disease mechanisms. However, challenges remain regarding throughput and coverage. A robust method is developed for high‐throughput proteoform imaging (HTPi) by combining matrix‐assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) and region‐specific top‐down proteomic analysis. MALDI MSI enables the imaging of proteoforms on tissue sections at a rate of 7 h cm−2 (100‐µm spatial resolution), and the identification sensitivity of the proteoforms is improved by narrow‐bore monolithic columns with low adsorption, yielding 366 annotated proteoform images from the mouse brain. The obtained proteoform images reveals differential expression of individual proteoforms across the brain regions, and distinct spatial distribution patterns of various proteoforms generated from a single gene. Given its ability to visualize proteoform, HTPi is further applied to explore spatial pathological changes associated with Alzheimer's disease (AD) in 5 × FAD mice. 158 annotated proteoform images are obtained in hippocampal regions at 50‐µm spatial resolution, illuminating 14 differential proteoforms in the subiculum region and highlighting their significant associations with amyloid‐β pathology in AD. The results highlight the power of HTPi in unraveling the intricate molecular landscape of brain tissues and its potential in elucidating disease mechanisms.