Chea Emily E, Rinas Aimee, Espino Jessica A, Jones Lisa M
Department of Pharmaceutical Sciences, University of Maryland Baltimore.
AIT Bioscience.
J Vis Exp. 2020 Mar 11(157). doi: 10.3791/60911.
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.
蛋白质快速光化学氧化法（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法。FPOP使用248纳米的准分子激光光解过氧化氢以产生羟基自由基。这些自由基会氧化修饰20种氨基酸中19种暴露于溶剂中的侧链。最近，该方法已应用于活细胞（IC-FPOP）中，以研究蛋白质在其天然环境中的相互作用。对细胞中蛋白质的研究考虑到了分子间拥挤以及体外研究中会被破坏的各种蛋白质相互作用。设计了一种定制的单细胞流动系统，以减少IC-FPOP过程中的细胞聚集和堵塞。该流动系统使细胞逐个通过准分子激光，从而确保一致的照射。通过比较FPOP产生的氧化程度与根据晶体结构计算出的蛋白质溶剂可及性，IC-FPOP可以准确探测蛋白质的溶剂可及侧链。
