Key Laboratory of Drug Metabolism and Pharmacokinetics, State Key Laboratory of Natural Medicines, College of Pharmacy, China Pharmaceutical University, Tongjiaxiang #24, Nanjing, Jiangsu 210009, China.
Analyst. 2019 Jan 14;144(2):512-520. doi: 10.1039/c8an01708e.
Native mass spectrometry has been recognized as a powerful tool for probing interactions between small molecules, such as drugs and natural products, and target proteins. However, the presence of heterogeneous proteins and metabolites in real biological systems can alter the conformations of target proteins or compete with candidate ligands, thus necessitating a method for measuring binding stoichiometries in matrices aside from the extensively used pure/recombinant protein systems. Furthermore, some small molecule-protein interactions have a transient and low-affinity nature and thus can be mis-assigned as nonspecific binding complexes that are often formed during the native ESI process. A native-denatured exchange (NDX) approach was recently developed using a reactive desorption electrospray ionization-mass spectrometer (DESI-MS) setup to screen specific interacting partners. The method works by gradually increasing the composition of denaturing solvents contained in the DESI spray and thus conferring a switch from a native to denatured ionization environment. This change impairs three-dimensional structures of target proteins and disrupts specific ligand-protein interactions, leading to decreased holo/apo ratios. In contrast, ligand-protein complexes exhibiting different trends are assigned as nonspecific interactions. Herein, we applied the NDX approach to probe specific ligand-protein interactions in biological matrices. We first used mixtures of model ligands and proteins to examine the use of reactive DESI-MS in recognizing ligand-target binding in mixtures. Subsequently, we used the NDX approach to analyze binding affinity curves of ligands to target proteins spiked in cell lysates with the aid of size exclusion chromatography and demonstrated its use in probing specific ligand-protein interactions from cell matrices.
天然质谱已被公认为一种强大的工具，可用于探测小分子（如药物和天然产物）与靶蛋白之间的相互作用。然而，真实生物体系中存在的异质蛋白质和代谢物会改变靶蛋白的构象，或与候选配体竞争，因此需要一种方法来测量除广泛使用的纯/重组蛋白体系之外的基质中的结合计量比。此外，一些小分子-蛋白质相互作用具有瞬态和低亲和力的性质，因此可能被错误地分配为非特异性结合复合物，这些复合物通常在天然 ESI 过程中形成。最近开发了一种天然变性交换 (NDX) 方法，使用反应性解吸电喷雾电离质谱仪 (DESI-MS) 装置来筛选特定的相互作用伙伴。该方法通过逐渐增加 DESI 喷雾中包含的变性溶剂的组成来工作，从而实现从天然到变性电离环境的转变。这种变化会破坏靶蛋白的三维结构并破坏特定的配体-蛋白质相互作用，导致全酶/脱辅基酶比值降低。相比之下，表现出不同趋势的配体-蛋白质复合物被分配为非特异性相互作用。在此，我们应用 NDX 方法来探测生物基质中的特定配体-蛋白质相互作用。我们首先使用模型配体和蛋白质的混合物来检查反应性 DESI-MS 在识别混合物中配体-靶标结合中的应用。随后，我们使用 NDX 方法分析了配体与细胞裂解物中添加的靶蛋白的结合亲和力曲线，借助尺寸排阻色谱法证明了其在从细胞基质中探测特定配体-蛋白质相互作用的用途。
