Electromobility Focusing for Separation of Proteins

用于蛋白质分离的电动聚焦

• 批准号: 7013144
• 负责人: MILTON L. LEE
• 金额: $ 35万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7013144
• 关键词: alpha fetoprotein; biomarker; blood chemistry; blood proteins; carcinoembryonal antigen; chorionic gonadotropin; clinical research; electrofocusing; human tissue; prostate specific antigen; protein purification; serology /serodiagnosis; technology /technique development; thymidine kinase

Protein distributions in biological specimens, such as body fluids, are reflective of the overall health of individuals. In addition, the presence or absence of specific marker proteins is indicative of certain disease states, such as cancer and heart disease. Determination of specific marker proteins in complex biological samples requires the use of analytical techniques with extremely high resolving power. Typical analyses are complicated by the wide dynamic range of protein concentrations in samples to be analyzed, which can exceed ten orders of magnitude. Often, abundant proteins interfere with the detection of trace target proteins. The overall objective of this proposal is to exploit the unique properties of a relatively new analytical technique called electromobility focusing or electric field gradient focusing (EFGF) for separating proteins in complex mixtures. The specific aims are (1) to optimize the pore structure of a protein-resistant polymeric monolith for minimizing flow dispersion in a dual-EFGF system and providing high resolution size exclusion chromatography (SEC) of proteins; (2) to serially integrate EFGF and SEC with UV detection and mass spectrometry; and (3) to demonstrate the performance of the protein analyzer for target protein analysis. New polymer chemistries discovered during the past contract period will be optimized and utilized for constructing EFGF devices for isolation of target proteins. A major advantage of EFGF is that trace target proteins can be isolated and concentrated while, at the same time, interfering proteins can be excluded from the separation system. The new analytical system will be demonstrated for detection of prostate specific antigen (PSA) as a target protein in its free and complexed forms.

生物样本（如体液）中的蛋白质分布反映了人类的整体健康状况。 个体此外，特异性标记蛋白的存在或不存在也是 指示某些疾病状态，例如癌症和心脏病。特异性标志物测定 复杂生物样品中的蛋白质需要使用分析技术， 分辨力典型的分析由于蛋白质的宽动态范围而变得复杂 待分析的样品中的浓度，其可以超过十个数量级。通常，丰富的 蛋白质干扰痕量靶蛋白的检测。本建议的总体目标是 利用一种称为电迁移率聚焦的相对较新的分析技术的独特性能 或电场梯度聚焦（EFGF）用于分离复杂混合物中的蛋白质。具体 目的是（1）优化抗蛋白质聚合物整料的孔结构， 在双EFGF系统中流动分散，并提供高分辨率尺寸排阻色谱 (SEC)（2）将EFGF和SEC串联集成于紫外检测器和质谱仪中; 以及（3）证明蛋白质分析仪用于目标蛋白质分析的性能。新 在过去的合同期内发现的聚合物化学品将得到优化和利用， 构建用于分离靶蛋白的EFGF装置。EFGF的一个主要优点是， 可以分离和浓缩靶蛋白，同时， 被排除在隔离系统之外。新的分析系统将用于检测 前列腺特异性抗原（PSA）以其游离和复合形式作为靶蛋白。