Supercritical Fluid Extraction for Membrane Proteomics

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The goal of this proposal is to develop a new approach to the mass spectrometric analysis of membrane, proteins. The proposed research addresses the critical problem with current membrane proteomics studies: namely, the efficient extraction of membrane proteins from biological samples in a form suitable for chromatography and high-throughput mass-spectrometrie analysis. Membrane proteins are typically solubilized in detergents: however, detergent solutions are not amenable to mass spectrometric analysis. This has presented a tremendous obstacle to the mass spectrometric analysis of membrane proteins. We propose to address this problem by using supercritical carbon dioxide as a mass spec- compatible solvent for membrane proteins. This solvent is a fluid with excellent dissolving power and complete transparency to a mass spectrometer. The work will proceed in two phases: in the first phase the basic feasibility of the approach will be evaluated by conducting studies on synthetic peptides and commercially available membrane proteins. The goal will be to demonstrate that these hydrophobic molecules can be solubilized in supercritical CO2, and that once solubilized they can be mass analyzed. In the second phase of the work the solubilized samples will be separated by supercritical fluid chromatography (SFC), a well-established method capable of fast separation with high resolution for non-polar compounds. This separation capability is critical to the analysis of complex mixtures. The approach will then be extended to the analysis of more complex biological samples such as a purified PhotoSystem I complex containing approximately 13 proteins and Fibroblast Growth Factor (FGF) receptor expressed in animal cell lines. The successful development of this new technology for membrane proteomics will address a critical gap in current proteomics efforts.