ITP-Based Selective Enrichment of Low Abundance Proteins

描述(由申请人提供)： 综合蛋白质组分析目前面临的最大挑战可能与蛋白质相对丰度的巨大变化有关(&gt；6个数量级)，在哺乳动物系统中具有潜在的生物学意义。此外，哺乳动物蛋白质组学中的样本数量有限，范围从1,000-100,000个细胞，对应于0.1-10微克的总蛋白质含量。例如，激光捕获显微切割技术的使用产生了以立方微米为单位的组织体积和亚微克范围的样本大小。此外，细胞和组织的异质性也有助于分析有限的亚群。 该项目的R43阶段旨在开发和展示基于瞬时毛细管等速/区带电泳(CITP/CZE)的多维分离平台，能够通过电动堆积(400-5000倍)提供显著的分析物浓度和对肽和蛋白质混合物的极高分辨率。最重要的是，在CITP中实现的分析物的最终浓度在很大程度上与领先缓冲液的摩尔比成正比。因此，CITP过程的结果是主要成分可能被稀释，但微量化合物被浓缩。这种对低丰度蛋白质的选择性增强可以大大缩小复杂蛋白质组中相对蛋白质丰度的范围，并利用所提出的基于CITP/CZE的多维分离技术大大提高蛋白质组的覆盖率。与置换层析相比，瞬时CITP/CZE具有速度快、在堆积(CITP)和分离(CZE)模式之间直接操作/切换的优点，并且不需要柱再生，包括去除结合置换体。 R43阶段的研究工作将致力于拟议的基于CITP/CZE的多维分离平台的早期实施、优化和演示，以用于酵母胰蛋白酶多肽的蛋白质组分析。R43阶段研究的结果将允许对技术进行验证，并提供必要的信息，以指导改进、设计和完成R44阶段研究中自动化、高通量、坚固、灵敏和超高分辨率的蛋白质组仪器。

DESCRIPTION (provided by applicant): Probably the greatest challenge presently facing comprehensive proteome analysis is related to the large variation of protein relative abundances (>6 orders of magnitude), having potential biological significance in mammalian systems. Additionally, limited sample amounts ranging from 1,000-100,000 cells are available in mammalian proteomics, corresponding to a total protein content of 0.1-10 micrograms. For example, the use of laser capture microdissection techniques yields tissue volumes in cubic micrometers and sample sizes in the sub-microgram range. Furthermore, the heterogeneous nature of cells and tissues also contributes to the requirement for analyzing limited subpopulations. The R43 Phase of this project aims to develop and demonstrate a transient capillary isotachophoresis/zone electrophoresis (CITP/CZE)-based multidimensional separation platform, capable of providing significant analyte concentration through electrokinetic stacking (400-5000 fold) and extremely high resolving power toward peptide and protein mixtures. Most importantly, the final concentration of the analyte achieved in CITP is largely proportional to the molarity of the leading buffer. Thus, the result of the CITP process is that major components may be diluted, but trace compounds are concentrated. 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/CZE-based multidimensional separation technology. In comparison with the displacement chromatography, the only reported approach in the literature to this point for the selective enrichment of low abundance peptides, transient CITP/CZE offers the benefits of speed, straightforward manipulation/switching between the stacking (CITP) and separation (CZE) modes, and no need for column regeneration, including the removal of bound displacers. Research efforts in the R43 Phase will address early implementation, optimization, and demonstration of the proposed CITP/CZE-based multidimensional separation platform for proteomic analysis of yeast tryptic peptides. The results of R43 Phase studies will allow validation of technology, and provide information necessary to guide the improvement, design, and completion of an automated, high throughput, robust, sensitive, and ultrahigh resolution proteome instrument in the R44 Phase studies.