A PCT Sample Preparation System for Proteomic Research and Clinical Diagnostics

用于蛋白质组学研究和临床诊断的 PCT 样品制备系统

基本信息

批准号：
7676137
负责人：
Alexander V Lazarev
金额：
$ 39.61万
依托单位：
PRESSURE BIOSCIENCE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-15 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7676137
关键词：
Adipose tissue Advanced Development Age Attention Basic Science Biochemical Biological Assay Biological Markers Biological Preservation Biological Process Biopsy Specimen Brain Buffers Carrier Proteins Cell Nucleus Cells Cellular Membrane Cellular Structures Chemicals Classification Clinical Collaborations Complex Cytoplasm Detergents Development Diabetes Mellitus Diagnostic Diagnostics Research Disease Drug Delivery Systems Enzyme-Linked Immunosorbent Assay Feasibility Studies Fractionation Future Goals Golgi Apparatus Health Heart Heart Diseases Histocompatibility Testing Hospitals Human Hydrostatic Pressure Image In Vitro Inner mitochondrial membrane Kidney Laboratories Left Lipid Peroxidation Lipids Liver Liver Mitochondria Lung Maintenance Manuals Measures Mediating Membrane Membrane Potentials Metabolic Methods Microsomes Mitochondria Mitochondrial Proteins Modification Morphology Muscle Myocardium Nucleic Acids Obesity Organelles Organic solvent product Outer Mitochondrial Membrane Oxidation-Reduction Oxidative Stress Phase Physiologic pulse Physiological Physiology Preclinical Drug Evaluation Preparation Process Property Proteins Proteome Proteomics Protocols documentation Reaction Reagent Recovery Reproducibility Research Resistance Respiration Rupture Sampling Skeletal Muscle Small Business Innovation Research Grant Staging Stroke Structure Subcellular structure System Technology Time Tissues Two-Dimensional Gel Electrophoresis Validation Western Blotting Woman animal tissue base clinical toxicology clinically relevant cost design drug discovery fluidity high standard human disease in vitro Assay in vivo metabolomics molecular marker new technology novel pressure research study tissue/cell culture

项目摘要

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.

描述（由申请人提供）：诸如线粒体等亚细胞成分的分离对于通过蛋白质组学和代谢组方法阐明其生物学功能至关重要。线粒体生理的异常已经与一系列疾病有关，包括中风，心脏病，糖尿病，肥胖和衰老。线粒体在现代研究中越来越受欢迎。除了作为潜在药物靶标的重要性外，功能性线粒体分离株对于高通量药物筛查至关重要。压力循环技术（PCT）使用受控的静水压力脉冲破坏膜和释放细胞成分。第一阶段的可行性实验表明，交替的压力可以是用来破坏细胞结构的一部分，同时使其留下完整。 PCT的这些选择性影响基于目标对压力快速变化的反应。已经表明，PCT参数的微调可用于隔离具有控制水平和可重现性的亚细胞结构，该结构优于传统方法。亚细胞蛋白质组学分析可以与通常集中在完整细胞或整个细胞裂解物的研究中互补。通过降低完整的细胞或组织的复杂性，根据其细胞定位的亚细胞成分的分离，分馏和浓度对于寻求低丰度蛋白生物标志物的寻求非常有用。该II阶段SBIR项目旨在开发可靠的平台技术，该技术将促进用于生物标志物研究，诊断和药物发现的完整和生物功能性亚细胞组件的隔离。我们的最初努力将放置在晚期PCT样品制备系统的发展中，以从培养的细胞和组织中隔离线粒体的需求。使用该系统将提取功能性线粒体以及其他显着性的细胞器。将对PCT方法进行优化研究，以最大程度地提高一系列临床相关组织类型的线粒体的产量和完整性 - 肝脏，肺，脑，心脏，心脏，骨骼肌和脂肪组织。可以预计，一旦细胞被PCT破裂，可以通过对线粒体提取优化的方案进行稍微修改，以有效地分离出线粒体的亚细胞成分，例如核或膜级分。此外，将研究一种基于PCT的亚线粒体分馏方法，以富集在特定线粒体室中局部的低丰度蛋白。努力将包括改进硬件，特定于样本容器的新设计，协议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，我们旨在提供商业平台，经过验证的协议和套件，以隔离动物组织的完整线粒体以及用户在将来的临床蛋白质组学诊断中提取人类术后样品和活检的优化方案。