Affinity Interactions in Capillary Separations

在这个由分析和表面化学计划支持的项目中，佛罗里达大学的罗伯特·肯尼迪教授和他的同事们将开发新的分析方法，使人们能够根据蛋白质与天然结合伙伴的亲和力来检测和量化蛋白质。在这些方法中，某些类别的蛋白质的荧光标记配体将被添加到样品中，生成的络合物将通过毛细管电泳法分离，并用激光诱导荧光进行检测。将被研究的结合系统包括用于检测G-蛋白的标记GTP、用于检测脱氢酶的NADH、用于检测含有src同源2结构域的蛋白的标记磷酸肽以及用于检测磷酸化蛋白的src同源2结构域。该方法有望提供高灵敏度(预计阿托莫尔检测下限)、高速(可能在秒到分钟的时间尺度上进行分析)和方便的自动化。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伙伴。由于生物系统中的信号转导广泛依赖于分子识别，这些亲和力方法有望在旨在揭示受体介导的细胞变化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，在那里它们最终可能有助于更好地了解胰岛素的分泌。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代码。然而，细胞并不是很容易定义的，即使是由它们制造的所有蛋白质。这些蛋白质的功能是什么？它们之间是如何互动的？如何控制相互作用和功能？对这些问题的简短回答是，蛋白质受它们的亲和力控制，即与细胞内选定的靶点或伙伴结合的能力。在这个项目中，寻求一种方法，允许基于蛋白质与特定分子的亲和力快速而灵敏地检测蛋白质。重要的是，该方法将允许分离和检测所有具有给定亲和力的蛋白质。新方法的灵敏度将至少是现有方法的1000倍，而且可以在几秒钟或几分钟内完成，而不是几个小时。这种新方法将允许对新的蛋白质或蛋白质混合物进行快速筛选，以确定其结合特性或功能。它还将允许对各种已知蛋白质进行快速检测。分析的速度和灵敏度将使其在药物发现、信号转导(即了解细胞内化学通讯)和生物技术等不同领域得到应用。

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.