Capillary sieving electrophoresis with a cationic surfactant for size-separations

使用阳离子表面活性剂进行毛细管筛分电泳进行尺寸分离

• 批准号: 7671047
• 负责人: Vladislav Dolnik
• 金额: $ 22.2万
• 依托单位: ALCOR BIOSEPARATIONS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7671047
• 关键词: Address; Biological Markers; Blood capillaries; Capillary Electrophoresis; Characteristics; Electrophoresis; Methods; Molecular Weight; Performance; Phase; Preparation; Protein Analysis; Proteins; Proteomics; Protocols documentation; Refractory; Reproducibility; Research Personnel; Resolution; Running; Sampling; Sodium Dodecyl Sulfate-PAGE; Speed; Technology; Time; Western Blotting; analytical method; capillary; gel electrophoresis; improved; method development; microchip; migration; molecular mass; public health relevance; quantum; surfactant; two-dimensional

DESCRIPTION (provided by applicant): The aim of this project is to develop and validate a fast, robust, reproducible, and high-resolution method of capillary sieving electrophoresis with a cationic surfactant for size-separations of proteins. Development of this method will represent a quantum leap in the proteomics technology bringing a fast, reproducible and fully automatable method for separation of proteins with unparalleled separation efficiency and reproducibility. The method will be ready for integration into automatable 2- dimensional capillary electrophoresis (2-D CE) of proteins. While automated 2-DE of proteins is highly desired by many protein researchers, cumbersome, laborious, and slow 2-dimensional slab gel electrophoresis remains a golden standard for protein separations. The proposed method has a strong potential to significantly contribute to the progress of proteomic technology and make search for protein biomarkers and target discoveries more efficient. The proposed method will also significantly improve accuracy of immunoanalysis and significantly reduce the analysis time when combined with the Western blot. Last but not least, the simple one- dimensional size separation and characterization of proteins. At high end, it will allow automated high throughput qualitative and quantitative analysis of a large number of samples in an array of capillaries and also high-speed analysis on a microchip. It is likely to address needs of a high number of researchers. In Phase I, we will develop and validate a method of capillary sieving electrophoresis with a cationic surfactant for one-dimensional quantitative analysis of proteins and their molecular weight determination. The method will have unmatched analytical characteristics, analyzing proteins in less than 12 minutes, with the separation efficiency exceeding 1,000,000 plates/m, and run-to-run reproducibility of migration times below 0.3%. In Phase I we will (1) optimize the composition of the sieving matrix, (2) validate CZECH as an analytical method for quantitative analysis of proteins, (3) validate CZECH as a method for molecular weight determination of proteins; (4) develop a protocol for sample preparation of refractory proteins to normalize their migration according to their molecular weights; (5) develop a Ferguson method to determine molecular mass of refractory proteins not responding to the modified sample preparation.. The proposed method will revolutionize protein analysis and enable advanced separation technologies that are otherwise impossible or difficult to perform. PUBLIC HEALTH RELEVANCE: The aim of this project is to develop and validate a fast, robust, reproducible, and high-resolution method of capillary sieving electrophoresis with a cationic surfactant for size-separations of proteins. The method will characterize separated proteins by determining their molecular weights in the range 3,500 - 400,000. Suppression of electroosmotic flow will allow separation efficiencies over one million theoretical plates/m and run-to-run reproducibility of migration times below 0.3%. The method will allow significantly improved 2-D separations of proteins.

描述（由申请人提供）：该项目的目的是开发和验证一种快速、稳健、可重复且高分辨率的毛细管筛分电泳方法，使用阳离子表面活性剂进行蛋白质尺寸分离。该方法的开发将代表蛋白质组学技术的巨大飞跃，带来一种快速、可重复且完全自动化的蛋白质分离方法，具有无与伦比的分离效率和重复性。该方法将准备好集成到蛋白质的自动化二维毛细管电泳（2-D CE）中。虽然许多蛋白质研究人员非常渴望蛋白质的自动化 2-DE，但繁琐、费力且缓慢的二维平板凝胶电泳仍然是蛋白质分离的黄金标准。所提出的方法具有极大的潜力，可以为蛋白质组学技术的进步做出重大贡献，并使蛋白质生物标志物的搜索和靶点发现更加有效。当与蛋白质印迹结合时，所提出的方法还将显着提高免疫分析的准确性并显着减少分析时间。最后但并非最不重要的一点是蛋白质的简单一维尺寸分离和表征。在高端，它将允许对毛细管阵列中的大量样品进行自动化高通量定性和定量分析，并在微芯片上进行高速分析。它可能会满足大量研究人员的需求。在第一阶段，我们将开发并验证一种使用阳离子表面活性剂的毛细管筛分电泳方法，用于蛋白质的一维定量分析及其分子量测定。该方法将具有无与伦比的分析特性，在不到 12 分钟的时间内分析蛋白质，分离效率超过 1,000,000 板/米，迁移时间的运行重复性低于 0.3%。在第一阶段，我们将 (1) 优化筛分基质的组成，(2) 验证 CZECH 作为蛋白质定量分析的分析方法，(3) 验证 CZECH 作为蛋白质分子量测定的方法； (4) 制定难熔蛋白的样品制备方案，根据其分子量标准化其迁移； (5) 开发一种弗格森方法来确定对改进的样品制备没有反应的难熔蛋白质的分子量。所提出的方法将彻底改变蛋白质分析，并使先进的分离技术成为可能，否则这些技术是不可能或难以执行的。公共健康相关性：该项目的目的是开发和验证一种快速、稳健、可重复且高分辨率的毛细管筛分电泳方法，使用阳离子表面活性剂对蛋白质进行尺寸分离。该方法将通过确定 3,500 - 400,000 范围内的分子量来表征分离的蛋白质。电渗流的抑制将使分离效率超过 100 万理论塔板/米，迁移时间的运行重复性低于 0.3%。该方法将显着改善蛋白质的二维分离。