Huo, S.; Ma, M.; Qian, S.; Zhang, M.; Pu, J.; Zhu, X.; Rasam, S.; Barone, T.; Plunkett, R.; Zhou, C.; Qu, J.

Whole-tissue spatial proteomics level provides critical insights into region-specific biological regulations but remains challenging. Previously, we introduced the Micro-scaffold Assisted Spatial Proteomics (MASP) concept for whole-tissue mapping. However, this prototype required substantial development in spatial resolution, practicality, and throughput for practical application. Here we present a next-generation MASP technique (hex-MASP) featuring i) a new design of hexagonal-micro-wells fabricated with optimized Projection Micro-Stereolithography (PuSL) 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. Applied to mouse brain, hex-MASP for the first time achieved in-depth, whole-tissue mapping for >6,000 proteins in mouse brains, with high spatial accuracy and excellent data quality. The substantially improved resolution revealed critical regional details across the entire brain, that were not previously captured, enabling precise depiction of protein distribution heterogeneity. This technique enabled the discovery of many unreported regionally-enriched proteins across brain structures. We further applied hex-MASP to investigate the intra-brain distribution of intracerebroventricularly-dosed antibody therapeutics and related proteins, which to our knowledge, enabled whole tissue mapping of protein drugs for the first time and revealed novel mechanistic insights into antibody distribution and localized treatment effects. Hex-MASP represent a robust, scalable platform for whole-tissue spatial proteomics.