Multiscale photocatalytic proximity labeling reveals cell surface neighbors on and between cells.

Proximity labeling proteomics (PLP) strategies are powerful approaches to yield snapshots of protein neighborhoods. Here, we describe a multiscale PLP method with adjustable resolution that uses a commercially available photocatalyst, Eosin Y, which upon visible light illumination activates different photo-probes with a range of labeling radii. We applied this platform to profile neighborhoods of the oncogenic epidermal growth factor receptor and orthogonally validated more than 20 neighbors using immunoassays and AlphaFold-Multimer prediction. We further profiled the protein neighborhoods of cell-cell synapses induced by bispecific T cell engagers and chimeric antigen receptor T cells. This integrated multiscale PLP platform maps local and distal protein networks on and between cell surfaces, which will aid in the systematic construction of the cell surface interactome, revealing horizontal signaling partners and reveal new immunotherapeutic opportunities.