López Abraham, Vilaseca Marta, Madurga Sergio, Varese Monica, Tarragó Teresa, Giralt Ernest
Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10, 08028, Barcelona, Spain.
Department of Organic Chemistry, University of Barcelona, Martí i Franquès 1, 08028, Barcelona, Spain.
J Mass Spectrom. 2016 Jul;51(7):504-11. doi: 10.1002/jms.3777.
Ion mobility mass spectrometry (IMMS) is a biophysical technique that allows the separation of isobaric species on the basis of their size and shape. The high separation capacity, sensitivity and relatively fast time scale measurements confer IMMS great potential for the study of proteins in slow (µs-ms) conformational equilibrium in solution. However, the use of this technique for examining dynamic proteins is still not generalized. One of the major limitations is the instability of protein ions in the gas phase, which raises the question as to what extent the structures detected reflect those in solution. Here, we addressed this issue by analyzing the conformational landscape of prolyl oligopeptidase (POP) - a model of a large dynamic enzyme in the µs-ms range - by native IMMS and compared the results obtained in the gas phase with those obtained in solution. In order to interpret the experimental results, we used theoretical simulations. In addition, the stability of POP gaseous ions was explored by charge reduction and collision-induced unfolding experiments. Our experiments disclosed two species of POP in the gas phase, which correlated well with the open and closed conformations in equilibrium in solution; moreover, a gas-phase collapsed form of POP was also detected. Therefore, our findings not only support the potential of IMMS for the study of multiple co-existing conformations of large proteins in slow dynamic equilibrium in solution but also stress the need for careful data analysis to avoid artifacts. Copyright © 2016 John Wiley & Sons, Ltd.
离子淌度质谱（IMMS）是一种生物物理技术，可根据等压物质的大小和形状对其进行分离。高分离能力、灵敏度以及相对快速的时间尺度测量赋予了IMMS在研究溶液中处于缓慢（微秒 - 毫秒）构象平衡的蛋白质方面的巨大潜力。然而，这项技术在检测动态蛋白质方面的应用尚未普及。主要限制之一是蛋白质离子在气相中的不稳定性，这就引发了一个问题，即检测到的结构在多大程度上反映了溶液中的结构。在此，我们通过天然IMMS分析脯氨酰寡肽酶（POP）的构象态势（一种在微秒 - 毫秒范围内的大型动态酶模型）来解决这个问题，并将气相中获得的结果与溶液中获得的结果进行比较。为了解释实验结果，我们使用了理论模拟。此外，通过电荷减少和碰撞诱导去折叠实验探索了POP气态离子的稳定性。我们的实验在气相中揭示了两种POP，它们与溶液中处于平衡的开放和闭合构象高度相关；此外，还检测到了一种气相塌陷形式的POP。因此，我们的研究结果不仅支持了IMMS在研究溶液中处于缓慢动态平衡的大型蛋白质的多种共存构象方面的潜力，也强调了进行仔细数据分析以避免假象的必要性。版权所有© 2016约翰威立父子有限公司。
