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）堆积过程的结果是主要组分可能被稀释，但痕量化合物被浓缩。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内相对蛋白质丰度的范围，并使用基于CITP的蛋白质组学技术大大提高蛋白质组覆盖率。因此，我们提出的研究工作的目的是充分表征，开发和利用这种差异浓度效应，以实现全面的蛋白质组分析的临床标本有限的样品可用性。激光捕获显微切割（LCM）过程提供了一种快速和直接的方法，用于分离选定的细胞亚群，用于下游生物化学和分子分析。在细胞富集的基础上，LCM还可作为一种靶向样品分级方法，以降低蛋白质复杂性和相对丰度。所提出的用于患病细胞富集的组织显微切割与基于CITP的选择性分析物浓缩的耦合不仅提供了用于检测和表征低丰度蛋白质的协同策略，而且还提供了新的生物标志物发现范例，用于能够鉴定肿瘤相关标志物，探索不同肿瘤状态和表型之间的分子关系，更深入地了解推动癌症进展的分子机制。 公共卫生相关性：通过将我们独特的生物分析能力与国家神经疾病和中风研究所（NINDS）的Zhengping Zhuang博士在组织显微切割和神经病理学方面的专业知识相结合，拟议的研究代表了一种协同努力，旨在开发，评估和验证一种新的生物标志物发现范式，用于支持癌症研究的肿瘤细胞及其微环境的蛋白质组学分析，诊断和治疗。

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.