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 (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.