Targeting Proteome Dynamic Range via Isotachophoresis-Based Analyte Enrichment

描述（由申请人提供）：该项目的R43阶段旨在开发和演示基于毛细管等速电泳（CITP）的多维蛋白质分离平台，能够提供选择性分析物富集和极高的分辨率，用于在质谱检测之前处理复杂的蛋白质混合物。这种对低丰度蛋白质的选择性增强可以大大降低复杂蛋白质组中相对蛋白质丰度的范围，并大大提高蛋白质组的覆盖率，使用所提出的CITP/纳米反相液相色谱（nano-RPLC）多维分离平台。拟议的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完整蛋白质分离，同时与当前和手动操作的蛋白质组学过程相比，避免了分析物的稀释和损失。通过与纳米级胰蛋白酶膜反应器耦合，从基于citp的多维分离中分离和洗脱的蛋白质的超快速蛋白水解消化可以在一次分析中直接将肽及其序列与相应的蛋白质和测量的蛋白质团块进行关联，并极大地促进了复杂蛋白质组翻译后修饰的表征。R43阶段的研究工作将涉及使用酵母细胞裂解物作为模型蛋白质组系统的蛋白质组技术的早期实施、优化和演示。

DESCRIPTION (provided by applicant): The R43 Phase of this project aims to develop and demonstrate a capillary isotachophoresis (CITP)-based multidimensional protein separation platform, capable of providing selective analyte enrichment and extremely high resolving power for handling complex protein mixtures prior to mass spectrometry detection. Such selective enhancement toward low abundance proteins can drastically reduce the range of relative protein abundances in complex proteomes, and greatly improve proteome coverage using the proposed CITP/nano-reversed-phase liquid chromatography (nano-RPLC) multidimensional separation platform. The proposed proteome technology will be highly automated and offer robust and high throughput fractionation of intact proteins for top-down proteomics while avoiding analyte dilution and loss in comparison with the current and manually operated proteome processes. By coupling with a nanoscale trypsin membrane reactor, the ultrafast proteolytic digestion of proteins resolved and eluted from the CITP-based multidimensional separations enables direct correlation of the peptides and their sequences with the corresponding proteins and measured protein masses in a single analysis, and greatly facilitates the 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 as a model proteome system.