Chromatographic Studies of Functional Proteomics

描述（由申请方提供）：糖尿病期间人血清白蛋白（HSA）的糖化被认为会影响该疾病期间药物的转运、置换和非结合血浆浓度。HSA是一种已知与多种药物相互作用的血液蛋白。当葡萄糖与HSA上的胺基发生非酶促反应形成稳定的果糖胺残基时，就会发生糖化，然后会产生其他产物。使用柱中固定的HSA的高效亲和色谱（HPAC）已被证明是用于研究药物-蛋白质相互作用的热力学、动力学和化学计量学的快速且精确的工具。HPAC在研究药物竞争、鉴定药物在HSA上的结合区域以及测量药物在其各种结合位点的缔合常数方面也是有价值的。我们最近表明，HPAC可以成为检查药物与糖化HSA相互作用的有效且强大的方法。在这个项目中，我们将寻求继续获得这样的信息，通过创建和采用高度新颖的HPAC检测和列，包含或检查糖化HSA和探索HPAC作为一种工具，用于检查异质蛋白质群体和复杂的生物系统中的相互作用。该项目的工作将通过三组可以并行进行的研究来完成。第一部分将寻求开发新的包埋相关技术的HPAC提供更快速和详细的信息之间发生的相互作用，药物和糖化HSA。这些方法将在本项目中得到证明和使用，以检查各种药物结合的变化和HSA糖基化后发生的药物-药物相互作用。第二部分将探讨新的HPAC方法的创建，可以提供一个直接和快速的评估药物在溶液中的蛋白质结合。这些方法将补充更详细的结合研究，使用包埋蛋白质，如在第一节中进行的，通过提供一种手段筛选溶液相蛋白质样品结合药物，并通过提供一种途径，可以很容易地实现与临床样品的使用。这些方法将被用于，特别是，检查药物-HSA相互作用的变化，发生在糖尿病患者之间和内部。第三部分将研究新的亲和/质谱方法的创建和使用，用于同时检查糖化HSA中发生的与药物相互作用相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，早期与晚期糖基化终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，因为它将更好地了解糖尿病如何影响HSA结合药物的能力， 其他化合物。此外，HPAC的方法和其他工具，在这个项目中开发的可用于在其他异质系统中的相互作用的研究，如与蛋白质，经历了不同程度的糖基化，磷酸化，或翻译后修饰的替代形式的工作。

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.