Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins
Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins
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DOI：
10.3791/60911
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发表时间：
2020-03-01
影响因子：
1.2
通讯作者：
Jones, Lisa M.
中科院分区：
文献类型：
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作者：
Chea, Emily E.;Rinas, Aimee;Jones, Lisa M.
蛋白质快速光化学氧化(FPOP)是一种用于表征蛋白质结构和相互作用的羟基自由基蛋白质足迹方法。FPOP使用248 nm准分子激光光解过氧化氢，产生羟基自由基。这些自由基氧化修饰20种氨基酸中19种的溶剂暴露侧链。最近，这种方法已经被用于活细胞(IC-FPOP)中，以研究其自然环境中的蛋白质相互作用。对细胞中蛋白质的研究解释了分子间拥挤和各种蛋白质相互作用，这在体外研究中受到了干扰。为减少IC-FPOP过程中的细胞聚集和堵塞，设计了一种定制的单细胞流动系统。这种流动系统使细胞分别聚焦通过准分子激光，从而确保持续的照射。通过将FPOP产生的氧化程度与根据晶体结构计算出的蛋白质的溶剂可及性进行比较，IC-FPOP可以准确地探测蛋白质的溶剂可及性侧链。
Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein's solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.