Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(hydroxymethyl)aminomethane

Fast photochemical oxidation of proteins (FPOP) is a powerful covalent labeling tool that uses hydroxyl radicals generated by laser flash photolysis of hydrogen peroxide to footprint protein surfaces. Because radical production varies with many experimental parameters, hydroxyl radical dosimeters have been introduced to track the effective radical dosage experienced by the protein analyte. FPOP experiments performed using adenine optical radical dosimetry containing protein in Tris buffer demonstrated unusual dosimetry behavior. We have investigated the behavior of Tris under oxidative conditions in detail. We find that Tris can act as a novel gain-of-signal optical hydroxyl radical dosimeter in FPOP experiments. This new dosimeter is also amenable to inline real-time monitoring, thereby allowing real-time adjustments to compensate for differences in samples for their quenching ability.