Diethylpyrocarbonate (DEPC)-based covalent labeling together with mass spectrometry is a promising tool for the higher-order structural analysis of antibody therapeutics. Reliable information about antibody higher-order structure can be obtained, though, only when the protein's structural integrity is preserved during labeling. In this work, we have evaluated the applicability of DEPC reaction kinetics for ensuring the structural integrity of monoclonal antibodies (mAbs) during labeling. By monitoring the modification extent of selected proteolytic fragments as a function of DEPC concentration, we find that a common DEPC concentration can be used for different monoclonal antibodies in formulated samples without perturbing their higher-order structure. Under these labeling conditions, we find that the antibodies can accommodate up to four DEPC modifications without being structurally perturbed, indicating that multidomain proteins can withstand more than one label, which contrasts to previously studied single-domain proteins. This more extensive labeling provides a more sensitive measure of structure, making DEPC-based covalent labeling-mass spectrometry suitable for the higher-order structural analyses of mAbs.

中文翻译：

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焦碳酸二乙酯的单克隆抗体的共价标记/质谱：确保蛋白质结构完整性的反应动力学。

基于焦碳酸二乙酯（DEPC）的共价标记以及质谱法是对抗体治疗剂进行更高级结构分析的有前途的工具。但是，只有在标记过程中保留蛋白质的结构完整性时，才能获得有关抗体高阶结构的可靠信息。在这项工作中，我们评估了DEPC反应动力学在确保标记过程中确保单克隆抗体（mAbs）结构完整性的适用性。通过监测所选蛋白水解片段的修饰程度作为DEPC浓度的函数，我们发现可以将共同的DEPC浓度用于配制样品中的不同单克隆抗体，而不会干扰它们的高阶结构。在这些标签条件下，我们发现抗体可以容纳多达四个DEPC修饰而不会在结构上受到干扰，这表明多域蛋白可以承受一个以上的标记，这与之前研究的单域蛋白形成了鲜明的对比。这种更广泛的标记提供了对结构的更灵敏的测量，使得基于DEPC的共价标记质谱法适合于mAb的高阶结构分析。