A PCT Sample Preparation System for Proteomic Research and Clinical Diagnostics

描述(申请人提供)：分离亚细胞成分，如线粒体，对于用蛋白质组学和代谢组学方法阐明它们的生物学功能具有重要意义。线粒体生理异常与一系列疾病有关，包括中风、心脏病、糖尿病、肥胖和衰老。线粒体是现代研究中越来越受欢迎的靶标。除了作为潜在药物靶点的重要性外，功能性线粒体分离株在高通量药物筛选中可能是至关重要的。压力循环技术(PCT)使用受控的静液压力脉冲来破坏细胞膜并释放细胞成分。第一阶段的可行性实验表明，交流压力可以 用来破坏细胞结构的一部分，而保持其他部分完好无损。PCT的这些选择性效应是基于目标对压力快速变化的反应。结果表明，微调PCT参数可用于分离亚细胞结构，其可控性和重复性优于传统方法。亚细胞蛋白质组图谱可以补充通常集中在完整细胞或整个细胞裂解物上的研究。通过降低完整细胞或组织的复杂性，根据细胞定位分离、分离和浓缩亚细胞成分在寻找低丰度蛋白质生物标志物方面非常有用。这个第二阶段的SBIR项目旨在开发一种强大的平台技术，该技术将促进分离完整的、具有生物功能的亚细胞成分，用于生物标记物研究、诊断和药物发现。我们最初的努力将放在开发先进的PCT样品制备系统，以按需从培养的细胞和组织中分离完整的线粒体。功能线粒体以及其他有意义的细胞器将使用该系统提取。将对PCT方法进行优化研究，以最大限度地提高来自一系列临床相关组织类型-肝、肺、脑、心脏、骨骼肌和脂肪组织-的线粒体的产量和完整性。预计，一旦细胞被PCT破坏，除了线粒体以外的亚细胞成分，如核或膜组分，可以通过对优化的线粒体提取方案进行轻微修改而有效地分离出来。此外，还将研究基于PCT的亚线粒体分离方法，以富集定位于特定线粒体区段的低丰度蛋白质。工作将包括硬件改进、样品专用容器的新设计、协议验证以及使用一些有代表性的下游应用程序对提取的产品进行检查。在这个项目的最后，我们的目标是提供一个商业平台，验证从动物组织中分离完整线粒体的方案和试剂盒，并提供优化方案，供用户在未来的临床蛋白质组诊断中用于提取人类手术后样本和活检组织。

DESCRIPTION (provided by applicant): Isolation of subcellular components, such as mitochondria, is of significant importance for the elucidation of their biological function by proteomic and metabolomic methods. Anomalies of mitochondrial physiology have been implicated in a range of disorders, including stroke, heart disease, diabetes, obesity and ageing. Mitochondria are increasingly popular targets in modern studies. In addition to their importance as potential drug targets, functional mitochondrial isolates could be crucial in high-throughput drug screening. Pressure Cycling Technology (PCT) uses controlled hydrostatic pressure pulses to disrupt membranes and release cellular components. The feasibility experiments in Phase I, demonstrated that alternating pressure can be used to disrupt parts of the cellular structure while leaving others intact. These selective effects of PCT are based on the target's reaction to rapid changes in pressure. It has been shown that fine tuning of PCT parameters can be used to isolate subcellular structures with a level of control and reproducibility that is superior to that of traditional methods. Subcellular proteomic profiling can be complementary to studies that often focus on intact cells or whole cell lysates. By reducing the complexity of intact cells or tissues, the isolation, fractionation and concentration of subcellular components according to their cellular localization can be very useful in the quest for low abundance protein biomarkers. This Phase II SBIR project is designed to develop a robust platform technology that will facilitate the isolation of intact and biologically functional subcellular components for biomarker research, diagnostics and drug discovery. Our initial effort will be placed in a development of the advanced PCT sample preparation system for on-demand isolation of intact mitochondria from cultured cells and tissues. Functional mitochondria as well as other organelles of significance will be extracted using this system. Optimization studies of PCT methods will be carried out to maximize yield and integrity of mitochondria from a set of clinically relevant tissue types - liver, lung, brain, heart, skeletal muscle and adipose tissue. It is anticipated that, once the cells are ruptured by PCT, subcellular components in additional to mitochondria, such as nuclei or membrane fractions, may be isolated efficiently by slight modification of the protocols optimized for mitochondrial extraction. Furthermore, A PCT-based sub-mitochondrial fractionation methods will be studied for the enrichment of low abundance proteins localized in specific mitochondrial compartments. Efforts will include hardware improvement, new designs of sample-specific containers, validations of protocols, and examinations of extracted products using a number of representative downstream applications. At the conclusion of this project, we aim to offer a commercial platform, validated protocols and kits for isolations of intact mitochondria from animal tissues, and optimization protocols that users may employ for extracting human postsurgical samples and biopsies in future clinical proteomic diagnostics.