Fast Photochemical Oxidation and Footprinting of Proteins Via Trifluoromethyl Radical Chemistry
Date of Award
Fast photochemical oxidation of proteins (FPOP) is a useful tool in proteomics because of the ability for modifications to occur on the scale of microseconds which reduces the modifications to tertiary and quaternary structure allowing for more accurate labeling of the protein. Labels for FPOP are generated from various radicals in our experiments which include hydroxyl radicals and trifluoromethyl radicals. Hydroxyl radicals are easily generated by using an excimer laser (KrF laser, 248 nm) or a UV flash lamp (as a part of the Fox™ System) by the photolysis of hydrogen peroxide. Trifluoromethyl radicals, however, need hydroxyl radicals to be generated because at this time, a suitable photolytic precursor compound has not been found to photolyze in the proper conditions need during standard FPOP.
Trifluoromethyl radicals are desired in footprinting because it can label 18 out of the 20 common amino acids as well as it could be used to label glycoproteins or carbohydrate because it does not break apart the carbon sugar rings like hydroxyl radicals do. The generation of trifluoromethyl radicals under these conditions will allow for easier footprinting of complex mixtures of carbohydrates and proteins without sacrificing structural integrity. By having a useful way to mark both R-groups on amino acids and carbohydrates, this can be used to develop proteomics for different pharmaceuticals and proteins within the body and widen our knowledge in protein-drug interactions.
Radical dosimetry is an extremely useful tool in FPOP and can give the user a helpful approximation of oxidation of the protein in question. Experiments on pyromellitic acid (PMA) were performed to see if it was a suitable candidate compound for dosimetry by fluorometry. Because of the chemical’s composition, price, and ease of use, it seemed like a prime candidate for inline fluorometry dosimetry with FPOP. Experiments and data showed an unknown reaction that showed the chemical fluorescence activity after exposure to the laser without an oxidant present.
Morrow, Elaine, "Fast Photochemical Oxidation and Footprinting of Proteins Via Trifluoromethyl Radical Chemistry" (2022). Honors Theses. 2541.
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