Photoproximity Labeling from Single Catalyst Sites Allows Calibration and Increased Resolution for Carbene Labeling of Protein Partners In Vitro and on Cells

The cell surface proteome (surfaceome) plays a pivotal role in virtually all extracellular biology, and yet we are only beginning to understand the protein complexes formed in this crowded environment. Recently, a high-resolution approach (mu Map) was described that utilizes multiple iridium-photocatalysts attached to a secondary antibody, directed to a primary antibody of a protein of interest, to identify proximal neighbors by light-activated conversion of a biotin-diazirine to a highly reactive carbene followed by LC/MS (Geri, J. B.; Oakley, J. V.; Reyes-Robles, T.; Wang, T.; McCarver, S. J.; White, C. H.; Rodriguez-Rivera, F. P.; Parker, D. L.; Hett, E. C.; Fadeyi, O. O.; Oslund, R. C.; MacMillan, D. W. C. Science 2020, 367, 1091-1097). Here we calibrated the spatial resolution for carbene labeling using site-specific conjugation of a single photocatalyst to a primary antibody drug, trastuzumab (Traz), in complex with its structurally well-characterized oncogene target, HER2. We observed relatively uniform carbene labeling across all amino acids, and a maximum distance of similar to 110 & Aring; from the fixed photocatalyst. When targeting HER2 overexpression cells, we identified 20 highly enriched HER2 neighbors, compared to a nonspecific membrane tethered catalyst. These studies identify new HER2 interactors and calibrate the radius of carbene photoprobe labeling for the surfaceome.