Cell Surface Engineering Enables Surfaceome ProfilingClick to copy article linkArticle link copied!
- Zak VilenZak VilenDepartment of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458, United StatesSkaggs Graduate School of Chemical and Biological Sciences, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United StatesDepartment of Molecular Medicine, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United StatesMore by Zak Vilen
- Abigail E. ReevesAbigail E. ReevesDepartment of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458, United StatesSkaggs Graduate School of Chemical and Biological Sciences, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United StatesDepartment of Molecular Medicine, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United StatesMore by Abigail E. Reeves
- Timothy R. O’LearyTimothy R. O’LearyDepartment of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458, United StatesMore by Timothy R. O’Leary
- Eugene JoehEugene JoehDepartment of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458, United StatesSkaggs Graduate School of Chemical and Biological Sciences, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United StatesDepartment of Molecular Medicine, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United StatesMore by Eugene Joeh
- Naomi KamasawaNaomi KamasawaThe Imaging Center and Electron Microscopy Core Facility, Max Planck Florida Institute for Neuroscience, 1 Max Planck Way, Jupiter, Florida 33458, United StatesMore by Naomi Kamasawa
- Mia L. Huang*Mia L. Huang*Email: [email protected]Department of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458, United StatesSkaggs Graduate School of Chemical and Biological Sciences, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United StatesDepartment of Molecular Medicine, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United StatesMore by Mia L. Huang
Abstract
Cell surface proteins (CSPs) are vital molecular mediators for cells and their extracellular environment. Thus, understanding which CSPs are displayed on cells, especially in different cell states, remains an important endeavor in cell biology. Here, we describe the integration of cell surface engineering with radical-mediated protein biotinylation to profile CSPs. This method relies on the prefunctionalization of cells with cholesterol lipid groups, followed by sortase-catalyzed conjugation with an APEX2 ascorbate peroxidase enzyme. In the presence of biotin-phenol and H2O2, APEX2 catalyzes the formation of highly reactive biotinyl radicals that covalently tag electron-rich residues within CSPs for subsequent streptavidin-based enrichment and analysis by quantitative mass spectrometry. While APEX2 is traditionally used to capture proximity-based interactomes, we envisioned using it in a “baitless” manner on cell surfaces to capture CSPs. We evaluate this strategy in light of another CSP labeling method that relies on the presence of cell surface sialic acid. Using the APEX2 strategy, we describe the CSPs found in three mammalian cell lines and compare CSPs in adherent versus three-dimensional pancreatic adenocarcinoma cells.
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This article is cited by 10 publications.
- Abigail E. Reeves, Zak Vilen, Trinity R. Fuentecilla, Christopher G. Parker, Mia L. Huang. Charting the Dynamic Trophoblast Plasma Membrane Identifies LYN As a Functional Regulator of Syncytialization. ACS Chemical Biology 2024, 19
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, 2220-2231. https://doi.org/10.1021/acschembio.4c00443
- Ting Wang, Yuying Liang, Guoli Wang, Shiyun Ma, Lei Zhang, Haojie Lu, Ying Zhang. Ultrafast and Chemoselective Biotinylation of Living Cell Surfaces for Time-Resolved Surfaceome Analysis. Analytical Chemistry 2024, 96
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, 2892-2904. https://doi.org/10.1016/j.ymthe.2024.07.019
- Nayara Braga Emidio, Ross W. Cheloha. Sortase-mediated labeling: Expanding frontiers in site-specific protein functionalization opens new research avenues. Current Opinion in Chemical Biology 2024, 80 , 102443. https://doi.org/10.1016/j.cbpa.2024.102443
- Xing Xu, Kejun Yin, Senhan Xu, Zeyu Wang, Ronghu Wu. Mass spectrometry-based methods for investigating the dynamics and organization of the surfaceome: exploring potential clinical implications. Expert Review of Proteomics 2024, 21
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, 99-113. https://doi.org/10.1080/14789450.2024.2314148
- Ryan R. Milione, Bin-Bin Schell, Cameron J. Douglas, Ciaran P. Seath. Creative approaches using proximity labeling to gain new biological insights. Trends in Biochemical Sciences 2024, 49
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- Abigail E. Reeves, Mia L. Huang. Proximity labeling technologies to illuminate glycan–protein interactions. Current Opinion in Chemical Biology 2023, 72 , 102233. https://doi.org/10.1016/j.cbpa.2022.102233
- Yansheng Zhai, Xiaoyan Huang, Keren Zhang, Yuchen Huang, Yanlong Jiang, Jingwei Cui, Zhe Zhang, Cookson K. C. Chiu, Weiye Zhong, Gang Li. Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling. Nature Communications 2022, 13
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https://doi.org/10.1038/s41467-022-32689-z
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