Allison Timothy M, Reading Eamonn, Liko Idlir, Baldwin Andrew J, Laganowsky Arthur, Robinson Carol V
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 5QY, UK.
Nat Commun. 2015 Oct 6;6:8551. doi: 10.1038/ncomms9551.
The effects of protein-ligand interactions on protein stability are typically monitored by a number of established solution-phase assays. Few translate readily to membrane proteins. We have developed an ion-mobility mass spectrometry approach, which discerns ligand binding to both soluble and membrane proteins directly via both changes in mass and ion mobility, and assesses the effects of these interactions on protein stability through measuring resistance to unfolding. Protein unfolding is induced through collisional activation, which causes changes in protein structure and consequently gas-phase mobility. This enables detailed characterization of the ligand-binding effects on the protein with unprecedented sensitivity. Here we describe the method and software required to extract from ion mobility data the parameters that enable a quantitative analysis of individual binding events. This methodology holds great promise for investigating biologically significant interactions between membrane proteins and both drugs and lipids that are recalcitrant to characterization by other means.
蛋白质 - 配体相互作用对蛋白质稳定性的影响通常通过一些既定的溶液相分析方法来监测。但很少有方法能轻易转化用于膜蛋白研究。我们开发了一种离子淌度质谱方法，该方法可通过质量和离子淌度的变化直接识别配体与可溶性蛋白和膜蛋白的结合，并通过测量对去折叠的抗性来评估这些相互作用对蛋白质稳定性的影响。通过碰撞激活诱导蛋白质去折叠，这会导致蛋白质结构变化，进而引起气相淌度变化。这使得能够以前所未有的灵敏度详细表征配体结合对蛋白质的影响。在此，我们描述了从离子淌度数据中提取参数所需的方法和软件，这些参数能够对单个结合事件进行定量分析。该方法对于研究膜蛋白与药物和脂质之间生物学上重要的相互作用具有巨大潜力，而这些相互作用很难通过其他方法进行表征。
