A PCT Sample Preparation System for Proteomic Research and Clinical Diagnostics

描述（由申请人提供）：亚细胞成分（如线粒体）的分离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也至关重要。压力循环技术（PCT）利用可控的静水压力脉冲破坏膜，释放细胞成分。第一期的可行性实验表明，交变压力是可行的

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.