CITP-Based Selective Tissue Proteome Enrichment

描述（由申请人提供）：与以相似程度普遍富集所有分析物相反，毛细管等速电泳（CITP）堆叠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将大大减少复杂组织蛋白质组中相对蛋白质丰度的范围，并极大地提高基于ccip的蛋白质组学技术的蛋白质组覆盖率。因此，我们提出的研究工作旨在充分表征、开发和利用这种差异浓度效应，在有限的样品可用性下实现临床标本的全面蛋白质组学分析。激光捕获显微解剖（LCM）过程提供了一种快速和直接的方法来分离选定的细胞亚群，用于下游生化和分子分析。在细胞富集的基础上，LCM还可以作为降低蛋白质复杂性和相对丰度的靶向样品分离方法。将病变细胞的组织显微解剖与基于ccip的选择性分析物浓度相结合，不仅为低丰度蛋白的检测和表征提供了协同策略，而且还为肿瘤相关标志物的鉴定、不同肿瘤状态和表型之间的分子关系的探索提供了一种新的生物标志物发现范式。以及对驱动癌症进展的分子机制的更深入了解。

DESCRIPTION (provided by applicant): In contrast to universally enriching all analytes by a similar degree, the result of the capillary isotachophoresis (CITP) stacking process is that major components may be diluted, but trace compounds are concentrated. Such selective enhancement toward low abundance proteins will drastically reduce the range of relative protein abundances within complex tissue proteomes, and greatly enhance the proteome coverage using the CITP-based proteomic technology. Our proposed research efforts therefore aim to fully characterize, develop, and exploit the use of this differential concentration effect to achieve comprehensive proteome analysis of clinical specimens with limited sample availability. The laser capture microdissection (LCM) process provides a rapid and straightforward method for isolating selected subpopulations of cells for downstream biochemical and molecular analyses. On the basis of cell enrichment, LCM also serves as a targeted sample fractionation approach toward the reduction in protein complexity and relative abundance. The proposed coupling of tissue microdissection for diseased cell enrichment with CITP-based selective analyte concentration not only presents a synergistic strategy for the detection and characterization of low abundance proteins, but also offers a novel biomarker discovery paradigm for enabling the identification of tumor-associated markers, exploration of molecular relationships among different tumor states and phenotypes, and a deeper understanding of molecular mechanisms that drive cancer progression. PUBLIC HEALTH RELEVANCE: By combining our unique bioanalytical capabilities with the expertise of Dr. Zhengping Zhuang at the National Institute of Neurological Disorders and Stroke (NINDS) in tissue microdissection and neuropathology, the proposed research represents a synergistic effort toward the development, evaluation and validation of a novel biomarker discovery paradigm for enabling the proteomic analysis of tumor cells and their micro-environment in support of cancer research, diagnosis, and treatment.