ITP-Based Selective Enrichment of Low Abundance Proteins

基于 ITP 的低丰度蛋白质选择性富集

基本信息

批准号：
6776989
负责人：
Brian M Balgley
金额：
$ 17.42万
依托单位：
CALIBRANT BIOSYSTEMS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-01 至 2006-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6776989
关键词：
capillary electrophoresis electrospray ionization mass spectrometry fungal proteins genome high throughput technology liquid chromatography molecular stacking protein quantitation /detection protein sequence proteomics reversed phase chromatography technology /technique development yeasts zonal electrophoresis

项目摘要

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.

描述（由申请人提供）：目前全面蛋白质组分析面临的最大挑战可能与蛋白质相对丰度的巨大变化（>6 个数量级）有关，这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可用的样品量有限，范围为 1,000-100,000 个细胞，相当于 0.1-10 微克的总蛋白质含量。例如，使用激光捕获显微切割技术可产生立方微米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阶段旨在开发和演示基于瞬态毛细管等速电泳/区带电泳（CITP/CZE）的多维分离平台，能够通过动电堆积（400-5000倍）提供显着的分析物浓度和极高的分辨率肽和蛋白质混合物。最重要的是，CITP 中分析物的最终浓度很大程度上与先导缓冲液的摩尔浓度成正比。因此，CITP 过程的结果是主要成分可能被稀释，但痕量化合物被浓缩。这种针对低丰度蛋白质的选择性增强可以大大减少复杂蛋白质组中相对蛋白质丰度的范围，并使用所提出的基于 CITP/CZE 的多维分离技术极大地提高蛋白质组覆盖率。与置换色谱法（迄今为止文献中唯一报道的选择性富集低丰度肽的方法）相比，瞬态 CITP/CZE 具有速度快、堆积 (CITP) 和分离 (CZE) 之间操作/切换简单的优点。）模式，并且不需要柱再生，包括去除结合的置换剂。 R43 阶段的研究工作将解决所提议的基于 CITP/CZE 的多维分离平台的早期实施、优化和演示，该平台用于酵母胰蛋白酶肽的蛋白质组分析。 R43 阶段研究的结果将验证技术，并提供必要的信息来指导 R44 阶段研究中自动化、高通量、稳健、灵敏和超高分辨率蛋白质组仪器的改进、设计和完成。