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Reputable Mentor II
Reputable Mentor II
Braden T. Lobingier,1,8 Ruth Hu¨ ttenhain,2,3,4,8 Kelsie Eichel,5 Kenneth B. Miller,6 Alice Y. Ting,7 Mark von Zastrow,1,2,9,* and Nevan J. Krogan2,3,4,
Cell Volume 169, Issue 2, p350–360.e12, 6 April 2017
Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.

http://www.cell.com/cell/abstract/S0092-8674(17)30302-1
1 Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94158, USA 2 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA 3 Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA 4 J. David Gladstone Institutes, San Francisco, CA 94158, USA 5 Program in Biochemistry and Molecular Biology, University of California, San Francisco, San Francisco, CA 94158, USA 6 Thermo Fisher Scientific, San Jose, CA 95134, USA 7 Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, CA 94305, USA 8 Co-first author 9 Lead Contact
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