Spatial proteomics at whole-tissue level provides critical insights into region-specific biological regulations but remains challenging. Previously, we introduced the Micro-scaffold Assisted Spatial Proteomics (MASP) technique, permitting whole-tissue mapping. While promising, this prototype required substantial improvements in spatial resolution, practicality, and throughput for practical application. Here we present a next-generation MASP technique (MASP-II) featuring i) a new design of hexagonal-micro-wells fabricated with optimized Projection Micro-Stereolithography (PµSL) 3D-printing, achieving high spatial resolution, sampling robustness and mechanical strength for reproducibly compartmentalizing even tough tissues; ii) enhanced throughput/effectiveness in sample preparation and LC-MS analysis with high quantitative quality. Using MASP-II, we generated in-depth, whole-tissue maps for >6,000 proteins in mouse brains, with thoroughly validated mapping accuracy. The improved resolution of MASP-II revealed critical details that were previously absent in maps by its prototype, enabling precise depiction of protein distribution heterogeneity. This technique enabled the discovery of many novel regionally enriched proteins across brain structures. Applying MASP-II to study the intra-brain distribution of intracerebroventricularly-dosed antibody drugs and related proteins revealed novel insights into antibody distribution mechanisms and local effects induced by drug treatment.