CITP-Based Selective Tissue Proteome Enrichment

描述(申请人提供)：与所有分析物的浓缩程度相同，毛细管等速电泳法(CITP)堆积过程的结果是主要成分可能被稀释，但痕量化合物被浓缩。这种对低丰度蛋白质的选择性增强将大大缩小复杂组织蛋白质组内相对蛋白质丰度的范围，并利用基于CITP的蛋白质组技术极大地提高蛋白质组的覆盖率。因此，我们建议的研究努力旨在充分表征、开发和利用这种差异浓度效应来实现对有限样本的临床标本的全面蛋白质组分析。激光捕获显微切割(LCM)过程提供了一种快速而直接的方法来分离选定的细胞亚群，用于下游的生化和分子分析。在细胞浓缩的基础上，LCM也是降低蛋白质复杂性和相对丰度的一种有针对性的样品分离方法。组织显微解剖和基于CITP的选择性分析物浓缩相结合，不仅为低丰度蛋白的检测和表征提供了一种协同策略，而且为识别肿瘤相关标志物、探索不同肿瘤状态和表型之间的分子关系以及更深入地理解推动癌症进展的分子机制提供了一种新的生物标志物发现范式。 与公共卫生相关：通过将我们独特的生物分析能力与美国国家神经疾病与中风研究所(NINDS)庄正平博士在组织显微解剖和神经病理学方面的专业知识相结合，拟议的研究代表着一项协同努力，旨在开发、评估和验证一种新的生物标记物发现范式，使能够对肿瘤细胞及其微环境进行蛋白质组分析，从而支持癌症的研究、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.