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Description
Understanding spatial heterogeneity of proteoforms has great potential to unravel physiological and disease mechanisms. However, conventional proteomics often lacks spatial information or focuses on the protein-coding gene level, while existing methods for spatial proteoform analysis suffer from low throughput or limited coverage. These gaps hinder the exploration of spatially resolved proteoform-function relationships
In this work, we developed high-throughput proteoform imaging (HTPi), an integrative workflow combining matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) with deep annotation via region-specific top-down proteomics, using a custom-designed narrow-bore monolithic column to enhance sensitivity. HTPi achieved proteoform visualization at 20–100 μm spatial resolution and annotated 366 proteoform images in mouse brain tissues, revealing distributions of individual proteoforms across different brain regions and distinct spatial patterns of proteoforms from a single gene (e.g., six Pcp4 proteoforms).
Applied to 5×FAD mice, HTPi was used to explore proteoform perturbations in the hippocampus, cortex, thalamus, and striatum. Notably, HTPi uncovered Aβ proteoforms (1–38, 1–40, 1–42) localized to subiculum plaques and identified 14 differential proteoforms, including truncated Ubb and mitochondrial Ndufv3. The co-localization of truncated Ubb proteoforms with Aβ plaques suggested a link between Aβ accumulation and ubiquitin-proteasome dysfunction. These results highlighted HTPi’s ability to resolve proteoform-level spatial dynamics in Alzheimer’s disease pathogenesis.
By bridging high-throughput MALDI MSI with region-specific top-down proteomics, HTPi advances spatial proteomics, offering insights into molecular mechanisms and potential biomarkers for neurodegenerative diseases. Future applications may expand to 3D brain-wide proteoform mapping and clinical translation for disease diagnosis.
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