Department of Early Stage Pharmaceutical Development and Department of Protein Analytical Chemistry-2, Genentech, Inc. , South San Francisco, California 94080, United States.
Anal Chem. 2017 Jan 17;89(2):1049-1053. doi: 10.1021/acs.analchem.6b03908. Epub 2017 Jan 3.
Amide hydrogen exchange experiments measured by mass spectrometry have become commonplace to study protein structural dynamics; however, the underdetermined nature of these measurements render extraction of exchange rates unreliable at the level of individual peptides. This prevents orthogonal verification of results and severely limits interpretation of the data. This work describes an easy-to-implement empirical method to determine the change in an observed rate constant or the average change in multiple rate constants as compared to some reference condition. This allows direct empirical computation of the average protection factor (PF) for peptides in isolation requiring no knowledge of actual rate constants themselves. Benchmarking the method by comparison of average peptide PFs with site-resolved NMR-derived PFs demonstrates high reliability and accuracy. This empirical method provides the first universally reliable strategy for recovering subglobal structural physics from individual peptides and, in doing so, standardizes the hydrogen exchange experiments measured by bottom-up mass spectrometry (HX MS), simplifies interpretation, and facilitates clear communication of the results.
酰胺氢交换实验通过质谱测量已成为研究蛋白质结构动力学的常用方法；然而，这些测量的欠定性质使得在单个肽水平上提取交换率不可靠。这阻止了结果的正交验证，并严重限制了对数据的解释。这项工作描述了一种易于实施的经验方法，用于确定与某些参考条件相比，观察到的速率常数的变化或多个速率常数的平均变化。这允许直接经验计算肽的平均保护因子 (PF)，而无需了解实际的速率常数本身。通过比较平均肽 PF 与基于位点的 NMR 衍生 PF 来对该方法进行基准测试，证明了其高度的可靠性和准确性。这种经验方法为从单个肽中恢复亚全局结构物理提供了第一个普遍可靠的策略，并以此为基础，标准化了通过自上而下的质谱测量的氢交换实验 (HX MS)，简化了解释，并促进了结果的清晰交流。
