Update publication information. Spatially resolved proteomics is an emerging approach, however, it remains technically challenging due to the complexity of the tissue microsampling techniques and mass spectrometry analysis of nanoscale specimen volumes. Here, we describe an expansion proteomics method ProteomEx, a combination of tissue expansion with mass spectrometry-based proteomics, which enables quantitative assessment of the spatial variability of the proteome in mammalian tissues at ~125 µm lateral resolution equivalent to the tissue volume of 0.7 nL using manual microsampling without the need for a custom or special equipment. We validated and demonstrated the utility of ProteomEx for high-throughput large-scale proteomics profiling of biological tissues including brain, liver, and breast cancer. We further applied ProteomEx for identifying proteins associated with Alzheimer's disease in a mouse model by comparative proteomic analysis of brain subregions.