Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins
| Autor: | Danté T. Johnson, Anne Gershenson, Benjamin Punshon-Smith, Jessica A. Espino, Lisa M. Jones |
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| Rok vydání: | 2021 |
| Předmět: |
Materials science
General Immunology and Microbiology Protein footprinting Protein Conformation General Chemical Engineering General Neuroscience Proteins Context (language use) Hydrogen Peroxide Laser Photochemistry Ligand (biochemistry) Proteomics Photochemical Processes General Biochemistry Genetics and Molecular Biology Article law.invention Protein–protein interaction Incubators Protein structure law Humans Protein folding Oxidation-Reduction |
| Zdroj: | J Vis Exp |
| ISSN: | 1940-087X |
| Popis: | Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO(2) and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H(2)O(2) concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput. |
| Databáze: | OpenAIRE |
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