In‐Situ Spatial Visual Proteomics Enabled by Single‐Cell‐Type Proximity Biotinylation and Signaling Amplification

Abstract Mapping the spatial proteome signature within heterogeneous tissue microenvironment of clinical specimens offers unique insights into both physiological and pathological molecular phenotypes. However, to simultaneously visualize and discover these spatial proteome features is challenging. Herein, we introduce spatial visual proteomics (SVPro) which integrates single‐cell proximity biotinylation coupled with fluorescence signal amplification via the trifunctional probe. This probe uniquely combines naphthylamine‐mediated radical targeting, clickable fluorescence imaging, and biotinylation‐facilitated proteome capture, enabling all three within a single tissue section. Strategic PEGylation and multivalent fluorescent conjugation of these probes improves labeling and visualization efficiency and specificity. SVPro allows cell‐type proteomic profiling of neuronal subpopulations in distinct mouse brain regions and amyloid‐β plaques in mouse brain of Alzheimer's disease model. Notably, we identified and validated distinct marker proteins associated with amyloid‑β plaques deposited in different brain regions. SVPro therefore emerges as a robust and high‐resolution spatial proteomics approach for simultaneously visualizing the spatial architecture and proteome landscapes of intact tissue microenvironment.