Department of BioMolecular Sciences , University of Mississippi , Oxford , Mississippi 38677 , United States.
Department of Chemistry and Biochemistry , University of Mississippi , Oxford , Mississippi 38677 , United States.
J Am Soc Mass Spectrom. 2020 Feb 5;31(2):169-172. doi: 10.1021/jasms.9b00088. Epub 2019 Dec 18.
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.
快速光化学氧化蛋白质（FPOP）是一种强大的共价标记工具，它利用过氧化氢的激光闪光光解产生的羟基自由基来标记蛋白质表面。由于自由基的产生受许多实验参数的影响，因此引入了羟基自由基剂量计来跟踪蛋白质分析物所经历的有效自由基剂量。在含有蛋白质的 Tris 缓冲液中使用腺嘌呤光学自由基剂量计进行的 FPOP 实验表现出异常的剂量计行为。我们详细研究了氧化条件下 Tris 的行为。我们发现，Tris 可以在 FPOP 实验中充当新型信号增益的光学羟基自由基剂量计。这种新的剂量计也适用于在线实时监测，从而可以实时调整以补偿样品的淬灭能力差异。
