A 3D spatial proteomic map of the human pancreatic islet microenvironment.

The human pancreas is a structurally and functionally complex organ in which endocrine islets are embedded within an exocrine matrix. Despite advances in spatial omics, three-dimensional (3D) proteomic map of the human islet microenvironment remains lacking. Here, we present a 3D spatial proteomics workflow that integrates immunofluorescence imaging, laser capture microdissection, nanoPOTS processing, and LC-MS/MS to map the islet microenvironment at 50 μm resolution, achieving ~3,000 protein identifications with spatial fidelity. Unsupervised clustering analyses revealed four molecularly distinct microenvironments spanning the acinar-to-islet axis, including a previously underappreciated peri-islet ductal-stromal niche enriched with canonical collagens and other extracellular matrix (ECM) proteins. Spatial correlation analysis (linking abundances to relative distance from the islet center) revealed proteins with interesting, reversed correlation patterns between islet and acinar compartments, which were enriched with ECM and cytoskeletal components. Finally, we provide a publicly accessible interactive web-based platform, enabling integrated 3D visualization of the spatial proteome alongside immunofluorescence imaging. Collectively, this work establishes a proof-of-principle framework for studying spatial tissue proteomic profiles, advances our understanding of the islet microenvironment, and lays a potential foundation for future applications in disease research.