To achieve a deep quantitative proteomic profiling of whole tissue with the spatial localization preserved, we designed an untargeted spatial proteomics workflow with a sparse sampling strategy. We combined a computational approach and MS-based proteomics analysis to localize high-throughput proteome mapping within a tissue by microdissecting the slices into a series of parallel strips with different angles, that allow subsequent image reconstruction. We successfully reconstructed a whole-tissue mapping in a mouse brain, consisting of 8,645 protein distributions and expressions with a spatial resolution of approx. 300 μm.