Capillary sieving electrophoresis with a cationic surfactant for size-separations

描述（由申请人提供）：该项目的目的是开发和验证一种快速、可靠、可重复和高分辨率的毛细管筛分电泳方法，该方法使用阳离子表面活性剂用于蛋白质的大小分离。该方法的发展将代表蛋白质组学技术的一个巨大飞跃，为蛋白质分离提供了一种快速、可重复和完全自动化的方法，具有无与伦比的分离效率和可重复性。该方法将被整合到自动化的2维毛细管电泳（2- d CE）蛋白质。虽然自动化的蛋白质2-DE是许多蛋白质研究人员高度期望的，但繁琐、费力和缓慢的二维平板凝胶电泳仍然是蛋白质分离的黄金标准。该方法对蛋白质组学技术的发展具有重要的促进作用，可以使蛋白质生物标志物的搜索和靶点的发现更加有效。该方法与Western blot结合使用，可显著提高免疫分析的准确性，显著缩短分析时间。最后但并非最不重要的是，简单的一维尺寸分离和表征蛋白质。在高端，它将允许在毛细管阵列中对大量样品进行自动化高通量定性和定量分析，也可以在微芯片上进行高速分析。它很可能满足大量研究人员的需求。在第一阶段，我们将开发和验证一种用阳离子表面活性剂进行毛细管筛分电泳的方法，用于蛋白质的一维定量分析和分子量测定。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/m，迁移次数的运行对运行重现性低于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，根据其分子量使其迁移正常化；(5)开发一种Ferguson方法来测定对改性样品制备无反应的难熔蛋白的分子质量。所提出的方法将彻底改变蛋白质分析，并使其他不可能或难以执行的先进分离技术成为可能。公共卫生相关性：该项目的目的是开发和验证一种快速、可靠、可重复和高分辨率的毛细管筛分电泳方法，该方法使用阳离子表面活性剂用于蛋白质的大小分离。该方法将通过测定其分子量在3,500 - 400,000范围内来表征分离的蛋白质。抑制电渗透流将使分离效率超过100万理论板/m，运行-运行迁移时间的再现性低于0.3%。该方法将显著改善蛋白质的二维分离。

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.