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

High Resolution Image Download MS PowerPoint Slide 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 (μMap) 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 ∼110 Å 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.