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Photoproximity Labeling from Single Catalyst Sites Allows Calibration and Increased Resolution for Carbene Labeling of Protein Partners In Vitro and on Cells.
- Source :
-
ACS central science [ACS Cent Sci] 2023 Dec 26; Vol. 10 (1), pp. 199-208. Date of Electronic Publication: 2023 Dec 26 (Print Publication: 2024). - Publication Year :
- 2023
-
Abstract
- 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.<br />Competing Interests: The authors declare no competing financial interest.<br /> (© 2023 The Authors. Published by American Chemical Society.)
Details
- Language :
- English
- ISSN :
- 2374-7943
- Volume :
- 10
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- ACS central science
- Publication Type :
- Academic Journal
- Accession number :
- 38292613
- Full Text :
- https://doi.org/10.1021/acscentsci.3c01473