Zhao, S.; Tan, W.; Yiu, A.; Liu, T.; Guo, X.; Camara, G. A.; Tian, H.; Wang, Y.; Dong, G.; Sun, C.; Ding, J.; Patra, P.; White, A. D.; Rodriques, S. G.; Mitchener, L.; Zhou, H.; Guo, T.; Liu, Y.; Piatkevich, K. D.

Correlating nanoscale tissue architecture with unbiased molecular composition within the same specimen remains a fundamental challenge in spatial biology. Current spatial proteomics methods either lack the imaging resolution required to resolve nanoscale tissue architecture or achieve only limited proteome coverage, and untargeted approaches capable of deep protein identification have yet to be integrated with super-resolution imaging within a single workflow. Here, we present microProteomEx, an integrated platform that combines hydrogel-assisted tissue expansion with mass spectrometry-compatible fluorescence imaging, laser capture microdissection, and bottom-up proteomics to achieve simultaneous three-dimensional super-resolution imaging (effective lateral resolution down to ~47 nm on conventional diffraction-limited microscopes) and spatially resolved proteomics at a scalable lateral resolution of 29-100 um (0.02-0.28 nL volumetric resolution). By optimizing fixation, protein anchoring, and a secondary re-embedding strategy, we extend the method to mechanically resilient tissues, including mouse kidney and heart, as well as to formalin-fixed, paraffin-embedded clinical specimens. We demonstrate single-glomerulus and single-plaque proteomics (450-1,000 proteins per structure), resolve proteomic differences between malignant melanoma and giant congenital melanocytic nevus in a rare pediatric case, and characterize the morphology-resolved molecular heterogeneity of cored versus diffuse amyloid-beta plaques in a mouse model of Alzheimer's disease across two disease stages. microProteomEx establishes a broadly accessible framework for correlating tissue ultrastructure with deep spatial proteomics, with direct implications for disease biology, biomarker discovery, and precision medicine.