Affinity Interactions in Capillary Separations

在这个由分析和表面化学计划支持的项目中，佛罗里达大学的Robert Kennedy教授和同事将开发新的分析方法，该方法允许基于蛋白质对天然结合伴侣的亲和力来检测和定量蛋白质。 在这些方法中，将蛋白质类的荧光标记配体添加到样品中，所得复合物将通过毛细管电泳分离并用激光诱导荧光检测。 将被研究的结合系统包括标记的GTP以检测G蛋白，NADH以检测磷酸化酶，标记的磷酸肽以检测含有src同源2结构域的蛋白，以及src同源2结构域以检测磷酸化蛋白。 该方法预计将提供高灵敏度（阿托摩尔检测限预期），高速（分析秒到分钟的时间尺度是可能的）和易于自动化。 这些方法有望用于跟踪蛋白质的表达水平或发现新的蛋白质和蛋白质的结合配偶体。 由于生物系统中的信号转导广泛依赖于分子识别，因此这些亲和方法预计在旨在揭示细胞中受体介导的变化的调节的实验中特别有用。 这些方法将在胰腺β细胞上进行测试，最终可能有助于更好地了解胰岛素分泌。基因组测序为生命提供了蓝图。 蓝图实际上是细胞中所有蛋白质的编码。 然而，细胞并不容易被定义，即使是它们制造的所有蛋白质。 蛋白质的功能是什么？它们是如何相互作用的？如何控制交互和功能？ 对这些问题的简短回答是，蛋白质由其亲和力控制，即，结合细胞内选定靶标或伴侣的能力。 在该项目中，寻求一种方法，该方法允许基于蛋白质对特定分子的亲和力快速灵敏地检测蛋白质。 重要的是，该方法将允许分离和检测具有给定亲和力的所有蛋白质。 新方法的灵敏度至少是现有方法的1000倍，并且可以在几秒钟或几分钟内完成，而不是几小时。 这种新方法将允许快速筛选新蛋白质或蛋白质混合物的某些结合特性或功能。 它还将允许快速测定已知蛋白质的种类。 所述测定的速度和灵敏度将使得能够应用于不同领域，例如药物发现、信号转导（即，理解细胞内化学通讯）和生物技术。

In this project supported by the Analytical and Surface Chemistry Program, Professor Robert Kennedy and co-workers at the University of Florida will develop novel analytical methods that allow proteins to be detected and quantified based on their affinity for natural binding partners. In the methods, fluorescently-labeled ligands for classes of proteins will be added to samples and the resulting complexes will be separated by capillary electrophoresis and detected with laser-induced fluorescence. Binding systems that will be investigated include labeled GTP to detect G-proteins, NADH to detect dehydrogenases, labeled phosphopeptides to detect proteins containing src homology 2 domains, and src homology 2 domains to detect phosphorylated proteins. The method is expected to provide high sensitivity (attomole detection limits are expected), high speed (analysis on the second to minute time scale are possible) and facile automation. The methods are expected to be useful for tracking the expression level of proteins or for discovery of novel proteins and binding partners of proteins. As signal transduction in biological systems relies extensively on molecular recognition, these affinity methods are expected to be especially useful in experiments aimed at uncovering the regulation of receptor-mediated changes in cells. The methods will be tested on pancreatic beta-cells where they may ultimately be useful in developing a better understanding of insulin secretion The sequencing of the genome has provided a blueprint for life. The blueprint is actually the code for all of the proteins that are made in a cell. Cells are not readily defined however even by all of the proteins that they make. What are the functions of the proteins? How do they interact with each other? How are the interactions and functions controlled? A short answer to these questions is that proteins are controlled by their affinity i.e., ability to bind selected targets or partners within the cell. In this project, a method is sought that allows proteins to be rapidly and sensitively detected based on their affinity for a particular molecule. Importantly, the method will allow all the proteins with a given affinity to be separated and detected. The new method will be at least 1000 times as sensitive as existing methods and can be performed in seconds or minutes rather than hours. This new method will allow new proteins or protein mixtures to be rapidly screened for certain binding properties or functions. It will also allow classes of known proteins to be rapidly assayed. The speed and sensitivity of the assays will enable applications in diverse fields such as drug discovery, signal transduction (i.e., understanding intracellular chemical communication), and biotechnology.