Krogan - GPCR-APEX

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Spatially and temporally resolved protein interaction networks in living cells.

  • Organism: Human
  • Instrument: TSQ Quantiva
  • SpikeIn: No
Cells operate through protein interaction networks organized in space and in time. Here we describe an approach to resolve both dimensions simultaneously 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 down-regulation after prolonged activation.
Experiment Description
Beta-2-adrenergic receptor and delta opioid receptor were tagged with APEX2 and expressed in HEK293 cells. Cells were pre-incubated with biotin-phenol for 30 minutes, and receptors were activated during this period at different time points (0, 1, 3, 10 and 30 min) with specific agonists. APEX labeling was activated by adding hydrogen peroxide (H2O2) for a 30 second biotinylation ‘snapshot’. Subsequently, biotinylated proteins were purified on streptavidin agarose. To identify and quantify relative abundance changes in biotin-labeled proteins following agonist stimulation, we developed a multi-step workflow using quantitative mass spectrometry (MS). For MS, biotin-labeled proteins were identified by shotgun proteomics and quantified by targeted proteomics using selected reaction monitoring (SRM).
Created on 3/3/17, 11:38 AM