Yamamoto Tatsuya, Yamagaki Tohru, Satake Honoo
Bioorganic Research Institute, Suntory Foundation for Life Sciences, Kyoto 619-0284, Japan.
Mass Spectrom (Tokyo). 2019;8(2):S0082. doi: 10.5702/massspectrometry.S0082. Epub 2020 Feb 14.
Hydrogen/deuterium exchange (HDX) coupled with pepsin digestion is useful for rapidly analyzing the kinetic properties of small amounts of protein. However, the analysis of HDX by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is time-consuming due to a lack of dedicated software. Currently available software programs mainly calculate average mass shifts, even though the isotopic distribution width contains information regarding multiple protein conformations. Moreover, HDX reaction samples are typically composed of peptides that contain various numbers of deuterium atoms, which also hinders the rapid and comprehensive analysis of protein dynamics. We report here on the development of a software program "Scipas DX" that can be used to automatically analyze the hydrogen-deuterium isotopic distribution in peaks in HDX spectra and calculate the average number of atoms exchanged, the average deuteration ratio, the abundance ratio for exchanged atoms, and their fitted spectra with a high degree of accuracy within a few minutes. Analysis of the abundance ratio for exchanged atoms of a model protein, adenylate kinase 1, using Scipas DX indicate that the local structure at residues 83-106 and 107-117 are in a slow equilibrium, suggesting that these regions adopt multiple conformations that are involved in the stability and in switching between the active and inactive forms. Furthermore, precise HDX kinetics of the average deuteration ratio both confirmed the known induced conformations of two regions (residues 46-75 and 131-165) that are responsible for ligand binding and verified the novel structural dynamics of residues 107-117 and 166-196 following ligand binding to ligand-binding pockets 1 and 2, respectively. Collectively, these results highlight the usefulness and versatility of Scipas DX in MALDI-MS HDX-based analyses of protein dynamics.
氢/氘交换（HDX）结合胃蛋白酶消化可用于快速分析少量蛋白质的动力学特性。然而，由于缺乏专用软件，通过基质辅助激光解吸/电离（MALDI）质谱（MS）分析HDX非常耗时。目前可用的软件程序主要计算平均质量位移，尽管同位素分布宽度包含有关多种蛋白质构象的信息。此外，HDX反应样品通常由含有不同数量氘原子的肽组成，这也阻碍了对蛋白质动力学的快速全面分析。我们在此报告了一个软件程序“Scipas DX”的开发，该程序可用于自动分析HDX光谱中峰的氢-氘同位素分布，并在几分钟内高精度地计算交换的原子平均数、平均氘化率、交换原子的丰度比及其拟合光谱。使用Scipas DX对模型蛋白腺苷酸激酶1的交换原子丰度比进行分析表明，83-106位和107-117位残基的局部结构处于缓慢平衡状态，这表明这些区域采用多种构象，这些构象与稳定性以及活性和非活性形式之间的转换有关。此外，平均氘化率的精确HDX动力学既证实了负责配体结合的两个区域（46-75位和131-165位残基）的已知诱导构象，又验证了配体分别与配体结合口袋1和2结合后107-117位和166-196位残基的新结构动力学。总的来说，这些结果突出了Scipas DX在基于MALDI-MS HDX的蛋白质动力学分析中的实用性和多功能性。
