SPOTTER: Automated Tissue-Barcoding Platform for Spatial Proteomics and Phosphoproteomics

Spatial proteomics aims to characterize proteome in situ with regional resolution, linking molecular states to tissue architecture and microenvironments. While recent advances have expanded our access to spatially resolved proteomics, major barriers in scalability and throughput remain. Building on our prior SPOT (Spatial Proteomics through On-site Tissue-protein-labeling) framework1, we introduce SPOTTER, a customizable robotic platform that enables automated, micron-scale spatial barcoding directly on intact tissue sections. SPOTTER barcodes proteins by labeling tissue proteins in situ to encode spatial origin across the whole tissue section. This strategy enables unbiased whole-tissue proteome mapping and, for the first time, spatial phosphoproteome profiling from intact sections. Coupled with high-resolution LC-MS/MS, SPOTTER achieves deep proteome and phosphoproteome coverage while preserving histological integrity. By replacing labor-intensive laser capture microdissection workflows and low-plex antibody arrays, SPOTTER provides a scalable route to high-plex spatial proteomics, resolving spatially distinct regions within a single experiment.