A PCT Sample Preparation System for Proteomic Research and Clinical Diagnostics

描述（由申请人提供）：分离亚细胞组分，如线粒体，对于通过蛋白质组学和代谢组学方法阐明其生物学功能具有重要意义。线粒体生理学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目标。除了作为潜在药物靶点的重要性外，功能性线粒体分离物在高通量药物筛选中可能至关重要。压力循环技术（PCT）使用受控的流体静压脉冲来破坏膜并释放细胞成分。第一阶段的可行性实验表明，交变压力可以 用来破坏细胞结构的一部分而使其他部分保持完整。PCT的这些选择性作用是基于目标对压力快速变化的反应。已经表明，PCT参数的微调可以用于分离亚细胞结构，其控制水平和再现性上级传统方法。亚细胞蛋白质组分析可以补充研究，往往集中在完整的细胞或全细胞裂解物。通过降低完整细胞或组织的复杂性，根据亚细胞组分的细胞定位分离、分级分离和浓缩亚细胞组分在寻找低丰度蛋白质生物标志物中可能非常有用。该二期SBIR项目旨在开发一种强大的平台技术，该技术将有助于分离完整的和具有生物功能的亚细胞组分，用于生物标志物研究，诊断和药物发现。我们最初的努力将放在先进的PCT样品制备系统的发展，按需分离完整的线粒体从培养的细胞和组织。功能性线粒体以及其他重要的细胞器将使用该系统提取。将进行PCT方法的优化研究，以最大限度地提高一组临床相关组织类型（肝脏、肺、脑、心脏、骨骼肌和脂肪组织）中线粒体的产量和完整性。预期一旦细胞被PCT破裂，除了线粒体之外的亚细胞组分，例如细胞核或膜组分，可以通过轻微修改优化线粒体提取的方案来有效分离。此外，基于PCT的亚线粒体分级方法将被研究用于富集定位于特定线粒体区室的低丰度蛋白。 努力将包括硬件改进、样品专用容器的新设计、方案验证以及使用许多代表性下游应用程序对提取产品进行检查。在该项目结束时，我们的目标是提供一个商业平台，验证协议和试剂盒，用于从动物组织中分离完整的线粒体，以及优化协议，用户可以在未来的临床蛋白质组学诊断中用于提取人类手术后样本和活检。

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.