Fast Photochemical Oxidation of Proteins
To ensure that there is sufficient sample of protein to be oxidized and then analyzed, we employed a flow system instead of firing a single laser shot into a small volume of protein . In this fast photochemical oxidation of proteins (FPOP) setup, the protein, which is mixed with 15 mM H2O2, is passed through fused silica tubing, irradiated at a certain point, and collected. The laser pulse is introduced into a region of the tubing where the polyimide coating is removed to afford a UV transparent window. To ensure that no fraction of irradiated protein is oxidized a second time (receives a second pulse of light), the laser frequency can be carefully matched to the solvent flow rate and tubing diameter. Furthermore, by designing the flow so that @20% of the protein solution is not irradiated, one is confident that all oxidations are from the first pulse of light, leaving unreacted protein to serve as a reference point. This FPOP approach should allow one to capture a fast ''snapshot'' of the protein in solution. To observe the oxidations (Fig. 11.8) on the protein, one can load approximately 5 pmol (80 ng) of protein onto a small trap column and desalt the protein with water. The mixture of oxidized and unoxidized protein is then eluted into a mass spectrometer (e.g. QTOF), enabling analysis of the unmodified and modified protein as shown in Fig. 11.8.
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