Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry
Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry
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DOI：
10.1021/ac051294f
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发表时间：
2006-02-15
影响因子：
7.4
通讯作者：
Griffin, PR
中科院分区：
文献类型：
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作者：
Chalmers, MJ;Busby, SA;Griffin, PR
酰胺氢/氘交换是一种强有力的生物物理技术，用于探测配体相互作用引起的蛋白质动力学变化。该技术固有的低通量限制了其对药物筛选和先导化合物优化的影响。自动化增加了H/D交换的吞吐量，使其与药物发现工作兼容。在这里，我们描述了第一个全自动的H/D交换系统，提供高度可重复的H/D交换动力学从130毫秒到24小时。通过平行样品处理使吞吐量最大化，并且系统可以在没有用户干预的情况下一式三份地运行H/D交换测定。我们证明了该系统的效用，以区分核受体PPAR γ的配体结合域（LBD）的结构扰动后，结合完全激动剂和部分激动剂诱导。PPAR γ是格列酮的靶点，格列酮是用于治疗与II型糖尿病相关的胰岛素抵抗的药物。最近，已经表明，部分激动剂的过氧化物酶体增殖物激活受体γ具有胰岛素增敏特性，而没有几个与完全激动剂药物相关的副作用。为了进一步研究部分激动剂激活PPAR γ的机制，我们将我们的研究扩展到分析配体与PPAR γ/RXR α，LBD的异二聚体复合物的相互作用。为了便于分析大蛋白质复合物的H/D交换，我们用14.5-T傅立叶变换离子回旋共振质谱仪进行了实验，该质谱仪能够在ppb范围内准确测量质量。
Amide hydrogen/deuterium exchange is a powerful biophysical technique for probing changes in protein dynamics induced by ligand interaction. The inherent low throughput of the technology has limited its impact on drug screening and lead optimization. Automation increases the throughput of H/D exchange to make it compatible with drug discovery efforts. Here we describe the first fully automated H/D exchange system that provides highly reproducible H/D exchange kinetics from 130 ms to 24 h. Throughput is maximized by parallel sample processing, and the system can run H/D exchange assays in triplicate without user intervention. We demonstrate the utility of this system to differentiate structural perturbations in the ligand-binding domain (LBD) of the nuclear receptor PPAR gamma induced upon binding a full agonist and a partial agonist. PPAR gamma is the target of glitazones, drugs used for treatment of insulin resistance associated with type II diabetes. Recently it has been shown that partial agonists of PPAR gamma have insulin sensitization properties while lacking several adverse effects associated with full agonist drugs. To further examine the mechanism of partial agonist activation of PPAR gamma, we extended our studies to the analysis of ligand interactions with the heterodimeric complex of PPAR gamma/RXR alpha, LBDs. To facilitate analysis of H/D exchange of large protein complexes, we performed the experiment with a 14.5-T Fourier transform ion cyclotron resonance mass spectrometer capable of measuring mass with accuracy in the ppb range.