Suppressing allostery in epitope mapping experiments using millisecond hydrogen / deuterium exchange mass spectrometry.
Suppressing allostery in epitope mapping experiments using millisecond hydrogen / deuterium exchange mass spectrometry.
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DOI：
10.1080/19420862.2017.1379641
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发表时间：
2017
期刊：
影响因子：
5.3
通讯作者：
Wilson DJ
中科院分区：
文献类型：
--
作者：
Deng B;Zhu S;Macklin AM;Xu J;Lento C;Sljoka A;Wilson DJ
定位候选抗体与其抗原靶标之间的界面，即通常所说的表位映射，是发展治疗性单抗的关键组成部分。随着最近商用自动化系统的出现，氢/氢交换(HDX)正迅速成为工业界和学术界研究人员首选的绘制表位的工具。然而，这种方法有一个明显的缺点，因为它可能会被相互作用产生的‘变构’结构和动态变化所混淆，但发生在远离接触点的地方(S)。在这里，我们介绍了一种在表位映射实验中抑制变构效应的“动力学”毫秒HDX工作流程。该方法采用了以前推出的微流控设备，可以通过芯片上的胃酶消化和电喷雾电离实现毫秒级的HDX标记时间。“动力学”工作流程也不同于传统的基于HDX的表位映射，因为抗体在HDX标记的开始就被引入到抗原中。以肌红蛋白/抗肌红蛋白为模型系统，我们证明了在较短的“动力学”工作流程标记时间(即200毫秒)下，HDX信号在“真实”表位上已经完全发育，但在变构部位仍大大低于显著阈值。通过使用刚性传递变构算法的计算对接预测和变构建模来支持对‘真’表位的识别。
Localization of the interface between the candidate antibody and its antigen target, commonly known as epitope mapping, is a critical component of the development of therapeutic monoclonal antibodies. With the recent availability of commercial automated systems, hydrogen / deuterium eXchange (HDX) is rapidly becoming the tool for mapping epitopes preferred by researchers in both industry and academia. However, this approach has a significant drawback in that it can be confounded by ‘allosteric’ structural and dynamic changes that result from the interaction, but occur far from the point(s) of contact. Here, we introduce a ‘kinetic’ millisecond HDX workflow that suppresses allosteric effects in epitope mapping experiments. The approach employs a previously introduced microfluidic apparatus that enables millisecond HDX labeling times with on-chip pepsin digestion and electrospray ionization. The ‘kinetic’ workflow also differs from conventional HDX-based epitope mapping in that the antibody is introduced to the antigen at the onset of HDX labeling. Using myoglobin / anti-myoglobin as a model system, we demonstrate that at short ‘kinetic’ workflow labeling times (i.e., 200 ms), the HDX signal is already fully developed at the ‘true’ epitope, but is still largely below the significance threshold at allosteric sites. Identification of the ‘true’ epitope is supported by computational docking predictions and allostery modeling using the rigidity transmission allostery algorithm.