Targeting Proteome Dynamic Range via Isotachophoresis-Based Analyte Enrichment

描述(申请人提供)：该项目的R43阶段旨在开发和演示一个基于毛细管等速电泳法(CITP)的多维蛋白质分离平台，该平台能够提供选择性的分析物浓缩和极高的分辨率，用于在质谱仪检测之前处理复杂的蛋白质混合物。这种对低丰度蛋白质的选择性增强可以显著缩小复杂蛋白质组的相对蛋白质丰度范围，并利用所提出的CITP/纳米反相液相色谱(Nano-RPLC)多维分离平台极大地提高蛋白质组覆盖率。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、高通量的完整蛋白质分级，同时与当前和人工操作的蛋白质组过程相比，避免分析物稀释和损失。通过与纳米级的胰酶膜反应器相结合，从基于CITP的多维分离中分离和洗脱的蛋白质的超快蛋白水解酶使多肽及其序列能够在单一分析中与相应的蛋白质和测量的蛋白质质量直接相关，从而极大地促进了复杂蛋白质组中翻译后修饰的表征。R43阶段的研究工作将解决拟议的蛋白质组技术的早期实施、优化和演示，使用酵母细胞裂解物作为蛋白质组系统的模型。

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.