SBIR-STTR Award

Substantial improvements in public health are anticipated in the near future through advancements in proteomic research leading to improvements in diagnosis of human diseases. These advancements will require new tools and standardized methods for basic and applied proteomic research, and clinical diagnostics. At this time, sample preparation systems and methods being used in proteomics and clinical diagnostics, lack the necessary efficiency, accuracy or robustness to adequately elucidate the proteome such that prediction of susceptibility to disease or response to drug therapy is efficacious. Here, we propose to develop an improved system and standard methods to study the proteome based on Pressure BioSciences' Pressure Cycling Technology (PCT), a proven powerful technology in extracting molecules from cells and tissues. Milestones of this proposed study include 1) Extraction of proteins from research and clinically relevant samples to demonstrate the feasibility of safe, reproducible (accurate) and high quality protein extraction; 2) Extraction of high quality and quantitative biomarkers, and 3) Extraction of molecular complexes, such as organelles. Our ultimate goal is to develop and establish a comprehensive system with standard methods for the extraction of proteins based on Pressure Cycling Technology (PCT) to accelerate discoveries in proteomics. Mirrored to human genome project, proteomics research and application development leads new concentrated efforts aiming to improve public health by revealing human proteome and its correlation with human diseases. Since human proteome is very complex, improved, reliable, and sensitive technologies are required for data generation and analysis. This proposed study aims to develop Pressure Cycling Technology for extracting high quality and quantity of proteins for the proteomic research and clinical diagnostics that may be highly significant in accelerating proteomic research and clinical application development.

Thesaurus Terms:
Clinical Chemistry, Method Development, Proteomics, Subcellular Fractionation, Tissue /Cell Preparation Biomarker, Cell Autolysis, Cell Osmotic Pressure, Membrane Protein, Molecular Assembly /Self Assembly, Organelle, Protein Purification, Protein Quantitation /Detection, Protein Structure Function, Two Dimensional Gel Electrophoresis Animal Tissue, Immunologic Assay /Test

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.

Public Health Relevance:
This Public Health Relevance is not available.

Thesaurus Terms:
There Are No Thesaurus Terms On File For This Project.