Decoding Single-Cell Protein Interactomes with the Proximity Network Assay 2309978

Abstract Introduction Proteins on the cell surface do not act in isolation, rather, their spatial organization and physical interactions are key determinants of the cellular function. Methods Using DNA-barcoded antibodies and proximity-dependent ligation, PNA simultaneously measures the abundance, clustering, and colocalization of 155 surface proteins, generating nanoscale surface maps comprising ∼50,000 molecular positions per cell without the use of optics. Results This spatially resolved readout enables systematic analysis of membrane protein networks across thousands of cells. We demonstrate the utility of PNA by identifying the proxiome of the CD19 CAR receptor at steady state and revealing dynamic proteomic remodeling during tumor cell encounter, including key phenomena such as trogocytosis and cell—cell conjugate formation. By integrating spatial context with multiplex protein profiling at scale, PNA provides a powerful platform for protein interactomics, biomarker discovery, and mechanistic insights across immunology, oncology, and cell therapy research. Conclusion By integrating spatial context with multiplex protein profiling at scale, PNA provides a powerful platform for protein interactomics, biomarker discovery, and mechanistic insights across immunology, oncology, and cell therapy research. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)