Diethylpyrocarbonate-Based Covalent Labeling Mass Spectrometry of Protein Interactions in a Membrane Complex System.
Diethylpyrocarbonate-Based Covalent Labeling Mass Spectrometry of Protein Interactions in a Membrane Complex System.
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基于焦碳酸二乙酯的膜复杂系统中蛋白质相互作用的共价标记质谱分析。
DOI：
10.1021/jasms.2c00262
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发表时间：
2023
影响因子：
3.2
通讯作者：
Vachet,RichardW
中科院分区：
文献类型：
--
作者：
Pan,Xiao;Tran,Thomas;Kirsch,ZacharyJ;Thompson,LynmarieK;Vachet,RichardW
膜相关蛋白是重要的，因为它们介导细胞的外部和内部环境之间的相互作用，并且它们通常是治疗的靶点。然而，表征它们的结构和结合相互作用是具有挑战性的，因为它们通常必须使用人工膜系统溶解，这可能使测量变得困难。质谱（MS）是研究膜相关蛋白质的一种重要工具，共价标记MS具有独特的潜力，可以提供复杂膜系统中这些蛋白质的高级结构和结合信息。在这里，我们证明，焦碳酸二乙酯（DEPC）可以有效地用作标记试剂，以表征在人工膜系统中的膜相关蛋白质和其结合伙伴之间的结合相互作用。使用趋化性组氨酸激酶（CheA）作为一个模型系统，我们表明，DEPC为基础的共价标记MS可以提供结构和结合信息的三元复合物的CheA与其他两种蛋白质，这是一致的结构模型，这种膜相关的化学感受器系统。尽管适度的疏水性DEPC，我们发现，它与蛋白质的反应性是没有实质性的人工膜的存在下的影响。然而，正确的结构信息，这种多蛋白质化学感受器系统需要测量DEPC标记在多个试剂浓度，使CheA和它的三元复合物的化学感受器系统之间的准确比较。除了提供与该复杂系统的模型一致的结构信息之外，标记数据补充了在化学感受器模型中没有充分细化的结构信息。
Membrane-associated proteins are important because they mediate interactions between a cell’s external and internal environment and they are often targets of therapeutics. Characterizing their structures and binding interactions, however, is challenging because they typically must be solubilized using artificial membrane systems that can make measurements difficult. Mass spectrometry (MS) is emerging as a valuable tool for studying membrane-associated proteins, and covalent labeling MS has unique potential to provide higher order structure and binding information for these proteins in complicated membrane systems. Here, we demonstrate that diethylpyrocarbonate (DEPC) can be effectively used as a labeling reagent to characterize the binding interactions between a membrane-associated protein and its binding partners in an artificial membrane system. Using chemotaxis histidine kinase (CheA) as a model system, we demonstrate that DEPC-based covalent labeling MS can provide structural and binding information about the ternary complex of CheA with two other proteins that is consistent with structural models of this membrane-associated chemoreceptor system. Despite the moderate hydrophobicity of DEPC, we find that its reactivity with proteins is not substantially influenced by the presence of the artificial membranes. However, correct structural information for this multiprotein chemoreceptor system requires measurements of DEPC labeling at multiple reagent concentrations to enable an accurate comparison between CheA and its ternary complex in the chemoreceptor system. In addition to providing structural information that is consistent with the model of this complex system, the labeling data supplements structural information that is not sufficiently refined in the chemoreceptor model.