CHROMATOGRAPHIC STUDIES OF FUNCTIONAL PROTEOMICS

描述（由申请人提供）：在糖尿病中，葡萄糖对人血清白蛋白（HSA）的非酶促修饰（称为糖化的过程）被认为影响药物的转运、置换和非结合蛋白浓度。 HSA 是一种血液蛋白，已知可与多种药物和其他溶质相互作用。过去的研究表明，使用固定化 HSA 柱的高效亲和层析 (HPAC) 是研究这些药物-蛋白质相互作用的热力学和化学计量的快速、精确的工具。然而，之前的研究尚未将这种方法用于糖化 HSA。这项工作的一个挑战是糖化 HSA 是一种异质蛋白质群体，其中糖化位点的数量和类型随血液中葡萄糖水平的变化而变化。此外，人们对这种糖基化如何影响 HSA 在该蛋白质特定区域的结合，或者这种修饰如何与糖尿病期间血液中的其他变化（例如脂肪酸水平增加，从而改变与 HSA 的药物相互作用）进行比较知之甚少。该项目的总体目标是开发和测试通过 HPAC 分析糖化 HSA 和其他异质配体系统的新方法。这将通过三组研究来完成。第一组将考虑使用 HSA/糖化 HSA 的包埋或位点选择性固定，以消除或最小化固定化引起的异质性，当这些蛋白质用于 HPAC 柱中以研究糖化 HSA 的固有异质性时；这项工作将使用从糖尿病患者样本中分离出的糖化 HSA 和体外糖化 HSA。第二组研究将利用色谱理论开发 HPAC 检测和表征配体结合异质性的程序。第三组研究将使用这些工具来检查 HSA 糖化时发生的药物相互作用的变化，并将这些影响与糖尿病中脂肪酸水平增加所产生的影响进行比较。该项目开发的工具也应可用于研究其他复杂配体的结合异质性，例如磷酸化或糖基化蛋白质。这项研究与公共健康相关，因为它将帮助我们更好地了解糖尿病如何影响 HSA 结合药物和其他化合物的能力。反过来，这应该可以更好地描述糖尿病患者和非糖尿病患者之间药物的转运、代谢和有效剂量的差异。

DESCRIPTION (provided by applicant): In diabetes, the non-enzymatic modification of human serum albumin (HSA) by glucose (a process known as glycation) is believed to effect the transport, displacement and non-bound protein concentrations of drugs. HSA is a blood protein known to interact with a wide range of drugs and other solutes. It has been shown in the past that high-performance affinity chromatography (HPAC) using immobilized HSA columns is a fast, precise tool for studying the thermodynamics and stoichiometries of these drug-protein interactions. However, no previous studies have used this approach with glycated HSA. One challenge in such work is the fact that glycated HSA is a heterogeneous protein population, in which the number and types of glycation sites vary with the levels of glucose in blood. In addition, little is known about how this glycation affects the binding of HSA at specific regions on this protein or how this modification compares with the other changes in blood during diabetes, such as increased levels of fatty acids, that can alter drug interactions with HSA. The overall goal of this project is to develop and test new approaches for the analysis of glycated HSA and other heterogeneous ligand systems by HPAC. This will be accomplished through three sets of studies. The first set will consider the use of entrapment or site-selective immobilization for HSA/glycated HSA to eliminate or minimize immobilization-induced heterogeneity when these proteins are used in HPAC columns for studying glycated HSA's inherent heterogeneity; this work will use both glycated HSA isolated from diabetic patient samples and in vitro glycated HSA. The second set of studies will use chromatographic theory to develop procedures by which HPAC can detect and characterize the binding heterogeneity of ligands. The third set of studies will use these tools to examine the changes in drug interactions that occur upon the glycation of HSA and to compare these effects with those produced by increased fatty acid levels in diabetes. The tools developed in this project should also be useful in studying the binding heterogeneity of other complex ligands, such as phosphorylated or glycosylated proteins. This research is relevant to public health since it will provide a better understanding of how diabetes affects the ability of HSA to bind drugs and other compounds. This, in turn, should allow an improved description of how the transport, metabolism, and effective dose of a drug will differ between a diabetic patient and an individual without diabetes.