Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform
Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform
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DOI：
10.1021/acs.analchem.6b01650
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发表时间：
2016-06-21
影响因子：
7.4
通讯作者：
Griffin, Patrick R.
中科院分区：
文献类型：
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作者：
Cummins, David J.;Espada, Alfonso;Griffin, Patrick R.
氢/氘交换质谱联用技术是一种信息丰富的生物物理学方法，可用于蛋白质动力学的表征。差异HDX-MS的成功应用包括蛋白质配体结合的表征。单个差异HDX-MS数据集（蛋白质+/-配体）通常由40多个单独的HDX-MS实验组成。为了消除费力的样品手动处理，并尽量减少随机和严重错误，HDX-MS分析的自动化系统已成为许多实验室的常规。然而，自动化系统虽然不太容易出现由人类操作员引入的随机错误，但可能具有在没有适当检测的情况下未被注意到的系统错误。尽管自动（和手动）HDX-MS的应用已经变得普遍，但只有少数研究报告了HDX-MS实验性能的系统评价，并且没有报告描述了HDX-MS实验的跨中心比较。在这里，我们描述了一个自动化HDX-MS平台，该平台采用平行，双阱，双柱配置，已安装在两个远程实验室中。为了了解实验室内和实验室之间的系统性能，我们设计并完成了一个测试-重测重复性研究，用于在两个实验室（一个在佛罗里达，另一个在西班牙）中的每个实验室实施的差分HDX-MS实验。本研究提供了足够的数据进行实验室内和实验室间变异性评估。初步结果显示，在两个系统之一的系统运行顺序的影响。因此，重复了该研究，这一次的结论是实验条件成功复制，系统误差最小。
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein ligand binding. A single differential HDX-MS data set (protein +/- ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.