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Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2021-11-30 , DOI: 10.1021/jacs.1c07771
Lukáš Fojtík 1, 2 , Jan Fiala 1, 2 , Petr Pompach 1, 3 , Josef Chmelík 1, 2 , Václav Matoušek 4 , Petr Beier 5 , Zdeněk Kukačka 1 , Petr Novák 1
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Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin–hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.

中文翻译:

蛋白质的快速氟烷基化揭示了生物大分子的结构和动力学

蛋白质的共价标记与质谱法相结合已被确立为经典结构方法的补充技术,例如用于蛋白质结构测定的 X 射线、NMR 或低温电子显微镜 (Cryo-EM)。尽管当前的共价标记技术能够监测具有足够空间分辨率的蛋白质溶剂可及区域,但对替代性、不太复杂和廉价的方法的需求仍然很高。在这里,我们介绍了一种基于蛋白质快速氟烷基化 (FFAP) 的新共价标记方法。FFAP 使用由 Togni 试剂与抗坏血酸还原分解形成的氟烷基自由基以秒为单位标记蛋白质。通过监测天然马心脏无肌红蛋白/全肌红蛋白和人触珠蛋白-血红蛋白复合物的差异氨基酸修饰,证明了 FFAP 有效标记蛋白质的可行性。获得的数据证实,Togni 试剂介导的 FFAP 是一种有利的替代方法,用于在蛋白质足迹和蛋白质(及其复合物)的表位作图等应用中进行共价标记。数据可通过带有数据集标识符 PXD027310 的 ProteomeXchange 服务器访问。获得的数据证实,Togni 试剂介导的 FFAP 是一种有利的替代方法,用于在蛋白质足迹和蛋白质(及其复合物)的表位作图等应用中进行共价标记。数据可通过带有数据集标识符 PXD027310 的 ProteomeXchange 服务器访问。获得的数据证实,Togni 试剂介导的 FFAP 是一种有利的替代方法,用于在蛋白质足迹和蛋白质(及其复合物)的表位作图等应用中进行共价标记。数据可通过带有数据集标识符 PXD027310 的 ProteomeXchange 服务器访问。
更新日期:2021-12-15
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