Simplified proteomics approach to discover protein-ligand interactions
Simplified proteomics approach to discover protein-ligand interactions
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DOI：
10.1002/pro.2112
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发表时间：
2012-09-01
期刊：
影响因子：
8
通讯作者：
Park, Chiwook
中科院分区：
文献类型：
--
作者：
Chang, Youngil;Schlebach, Jonathan P.;Park, Chiwook
在药物研究和化学遗传学中，确定具有生物活性的小分子靶点是一项重要但仍然具有挑战性的任务。基于能量学的目标识别是一种利用配体结合时蛋白质构象稳定性的变化来识别目标蛋白质的方法。与传统的基于亲和力的捕获方法不同，基于能量学的方法不需要对测试分子进行任何标记或固定。在这里，我们报告了一种令人惊讶的基于能量学的目标识别的简单版本，它只需要离子交换色谱，SDS PAGE和少量使用质谱。通过离子交换色谱的分离，降低了蛋白质组的复杂性。然后通过在存在和不存在配体的情况下展开时蛋白水解敏感性的显着增加来监测每个部分中尿素诱导的蛋白质展开。与配体展开程度不同的蛋白质通过SDS - PAGE和质谱鉴定。使用这种方法，我们在大肠杆菌蛋白质组中鉴定了atp结合蛋白。除了已知的ATP结合蛋白，我们还鉴定了一些以前不知道与ATP相互作用的蛋白。为了验证这样的一个发现，我们克隆并纯化了磷酸甘油转化酶，这是以前不知道的与ATP结合，并证实ATP确实稳定这种蛋白质。分离和脉冲蛋白水解的结合提供了在蛋白质组学尺度上研究蛋白质药物或蛋白质代谢物相互作用的机会，使用最小的仪器，而不需要对感兴趣的分子进行修饰。
Identifying targets of biologically active small molecules is an essential but still challenging task in drug research and chemical genetics. Energetics-based target identification is an approach that utilizes the change in the conformational stabilities of proteins upon ligand binding in order to identify target proteins. Different from traditional affinity-based capture approaches, energetics-based methods do not require any labeling or immobilization of the test molecule. Here, we report a surprisingly simple version of energetics-based target identification, which only requires ion exchange chromatography, SDS PAGE, and minimal use of mass spectrometry. The complexity of a proteome is reduced through fractionation by ion exchange chromatography. Urea-induced unfolding of proteins in each fraction is then monitored by the significant increase in proteolytic susceptibility upon unfolding in the presence and the absence of a ligand. Proteins showing a different degree of unfolding with the ligand are identified by SDS PAGE followed by mass spectrometry. Using this approach, we identified ATP-binding proteins in the Escherichia coli proteome. In addition to known ATP-binding proteins, we also identified a number of proteins that were not previously known to interact with ATP. To validate one such finding, we cloned and purified phosphoglyceromutase, which was not previously known to bind ATP, and confirmed that ATP indeed stabilizes this protein. The combination of fractionation and pulse proteolysis offers an opportunity to investigate proteindrug or proteinmetabolite interactions on a proteomic scale with minimal instrumentation and without modification of a molecule of interest.