Integrated Top-Down/Bottom-Up Comprehensive Proteomics

描述(申请人提供)：第一阶段：该项目的R43阶段旨在开发和演示基于毛细管凝胶电泳(CGE)的多维蛋白质分离平台，该平台能够提供显著的分析物浓度和极高的分辨率，用于在质谱仪检测之前处理复杂的蛋白质混合物。与传统的基于SDS-PAGE的蛋白质组学方法相比，所提出的CGE/毛细管反相液相色谱(CRPLC)蛋白质组学技术将高度自动化，为自上而下的蛋白质组学提供可靠和高通量的完整蛋白质分离，同时避免在一个集成平台中分析物的稀释和损失。通过与纳米级的胰酶膜反应器相结合，从基于CGE的分离中分离和洗脱出来的蛋白质的超快蛋白分解消化使得能够对复杂蛋白质组中的翻译后修饰进行自上而下/自下而上的组合表征。R43阶段的研究工作将致力于使用酵母细胞裂解物对拟议的蛋白质组技术进行早期实施、优化和演示。

DESCRIPTION (provided by applicant): Phase I: The R43 phase of this project aims to develop and demonstrate a capillary gel electrophoresis (CGE) based multidimensional protein separation platform capable of providing significant analyte concentration and extremely high resolving power for handling complex protein mixtures prior to mass spectrometry detection. In comparison with conventional SDS-PAGE-based proteome approaches, the proposed CGE/capillary reversed-phase liquid chromatography (CRPLC) proteome technology will be highly automated and offer robust and high throughput fractionation of whole proteins for top-down proteomics while avoiding analyte dilution and loss in an integrated platform. By coupling with a nanoscale trypsin membrane reactor, the ultrafast proteolytic digestion of proteins resolved and eluted from the CGE-based separations enables the combined top-down/bottom-up characterization of post-translational modifications in complex proteomes. Research efforts in the R43 phase will address early implementation, optimization, and demonstration of the proposed proteome technology using yeast cell lysates.