Chromatographic Studies of Functional Proteomics

描述(由申请人提供)：糖尿病期间人血清白蛋白(HSA)的糖基化被认为影响药物在该病期间的转运、置换和非结合血浆浓度。人血清白蛋白是一种血液蛋白，已知可与多种药物相互作用。当葡萄糖与人血清白蛋白上的胺基团发生非酶反应，形成稳定的果糖胺残基，从而产生其他产物时，糖基化就发生了。使用固定化HSA的高效亲和层析(HPAC)已被证明是研究药物-蛋白质相互作用的热力学、动力学和化学计量的一种快速而精确的工具。HPAC在研究药物-药物竞争、确定药物在人血清白蛋白上的结合区域以及测量药物在其不同结合部位的结合常数方面也是有价值的。我们最近已经证明，HPAC可以成为一种有效和强大的方法来检查药物与糖化HSA的相互作用。在这个项目中，我们将寻求通过创建和使用包含或检查糖化HSA的高度新颖的HPAC分析和柱来继续获取此类信息，并探索HPAC作为检查异质蛋白质群体和复杂生物系统中相互作用的工具。该项目的工作将通过三组可以并行进行的研究来完成。第一部分将寻求为HPAC开发新的包埋相关技术，以提供关于药物和糖化HSA之间发生的相互作用的更快速和更详细的信息。这些方法将在这个项目中演示和使用，以检查各种药物结合的变化以及HSA糖基化时发生的药物-药物相互作用的变化。第二部分将探索创造新的HPAC方法，以提供对溶液中药物-蛋白质结合的直接和快速评估。这些方法将是对第一节中进行的使用包裹蛋白质进行的更详细的结合研究的补充，因为它们提供了一种筛选与药物结合的溶液相蛋白质样本的手段，并提供了一条易于实施的用于临床样本的路线。这些方法将特别用于检查糖尿病患者之间和患者内部发生的药物-人血清白蛋白相互作用的变化。第三部分将研究新的亲和/质谱学方法的创建和使用，以同时检查糖化人血清白蛋白中与药物相互作用有关的结构和功能变化。这些方法的一个具体应用将是提供更详细的信息，说明早期和晚期糖基化终末产物在影响药物与糖化HSA相互作用中所起的相对作用。这项研究与公共卫生有关，因为它将提供更好的了解糖尿病如何影响HSA结合药物和 其他化合物。此外，在本项目中开发的HPAC方法和其他工具可以用于研究其他不同体系中的相互作用，例如与经历了不同程度糖基化、磷酸化或其他形式的翻译后修饰的蛋白质的工作。

DESCRIPTION (provided by applicant): The glycation of human serum albumin (HSA) during diabetes is believed to affect the transport, displacement, and non-bound plasma concentrations of drugs during this disease. HSA is a blood protein known to interact with a wide range of drugs. Glycation occurs when glucose undergoes a non-enzymatic reaction with amine groups on HSA to form stable fructosamine residues, which can then lead to other products. High-performance affinity chromatography (HPAC) using immobilized HSA in columns has been shown to be a fast and precise tool for studying the thermodynamics, kinetics, and stoichiometries of drug-protein interactions. HPAC is also valuable in studying drug-drug competition, identifying the binding regions for a drug on HSA, and measuring association constants for a drug at its various binding sites. We have recently shown that HPAC can be an effective and powerful method for examining drug interactions with glycated HSA. In this project, we will seek to continue obtaining such information by creating and employing highly novel HPAC assays and columns that contain or examine glycated HSA and exploring HPAC as a tool for examining interactions in heterogeneous protein populations and complex biological systems. Work in this project will be accomplished through three sets of studies that can be conducted in parallel. The first section will seek to develop novel entrapment-related techniques for HPAC to provide more rapid and detailed information on the interactions that occur between drugs and glycated HSA. These methods will be demonstrated and used in this project to examine the changes in binding by various drugs and in drug-drug interactions that occur upon the glycation of HSA. The second section will explore the creation of new HPAC methods that can provide a direct and rapid assessment of drug-protein binding in solution. These methods will be complementary to the more detailed binding studies using entrapped proteins, as conducted in the first section, by providing a means for screening solution-phase protein samples for binding to drugs and by providing a route that can be easily implemented for use with clinical samples. These methods will be used, in particular, to examine the variations in drug-HSA interactions that occur between and within diabetic patients. The third section will examine the creation and use of new affinity/mass spectrometry methods for simultaneously examining the structural and functional changes that occur in glycated HSA as related to drug interactions. A specific application to be examined for these methods will be to provide more detailed information on the relative role played by early vs. advanced glycation end-products in affecting drug interactions with glycated HSA. This research is relevant to public health because it will provide a better understanding of how diabetes affects the ability of HSA to bind drugs and other compounds. In addition, the HPAC methods and other tools developed in this project could be employed in the study of interactions in other heterogeneous systems, such as work with proteins that have undergone various degrees of glycosylation, phosphorylation, or alternative forms of post-translational modification.