Integrated Top-Down/Bottom-Up Comprehensive Proteomics

描述（由申请人提供）：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，该平台能够在质谱检测之前提供显著的分析物浓度和极高的分辨率，以处理复杂的蛋白质混合物。与传统的基于sds - page的蛋白质组方法相比，所提出的CGE/毛细管反相液相色谱（CRPLC）蛋白质组技术将高度自动化，并为自上而下的蛋白质组学提供强大的高通量全蛋白分离，同时避免了分析物在集成平台中的稀释和损失。通过与纳米级胰蛋白酶膜反应器相结合，从基于cge的分离物中分离和洗脱的蛋白质的超快速蛋白质水解消化使得复杂蛋白质组中翻译后修饰的自上而下/自下而上的组合表征成为可能。R43阶段的研究工作将涉及使用酵母细胞裂解物的蛋白质组技术的早期实施、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.