Supercritical Fluid Extraction for Membrane Proteomics

描述（由申请人提供）： 本研究的目的是开发一种膜蛋白质质谱分析的新方法。拟议的研究解决了当前膜蛋白质组学研究的关键问题：即，从生物样品中有效提取膜蛋白，以适合色谱和高通量质谱分析的形式。膜蛋白通常溶解在去污剂中：然而，去污剂溶液不适合质谱分析。这给膜蛋白的质谱分析带来了巨大的障碍。我们建议通过使用超临界二氧化碳作为膜蛋白的质谱兼容溶剂来解决这个问题。该溶剂是具有优异溶解能力和对质谱仪完全透明的流体。这项工作将分两个阶段进行：在第一阶段，将通过对合成肽和商业上可获得的膜蛋白进行研究来评估该方法的基本可行性。目标是证明这些疏水分子可以溶解在超临界CO2中，并且一旦溶解，它们可以进行质量分析。在工作的第二阶段，溶解的样品将通过超临界流体色谱法（SFC）分离，这是一种能够快速分离非极性化合物的成熟方法。这种分离能力对于复杂混合物的分析至关重要。然后，该方法将扩展到更复杂的生物样品的分析，如纯化的光系统I复合物，含有约13种蛋白质和动物细胞系中表达的成纤维细胞生长因子（FGF）受体。这种膜蛋白质组学新技术的成功开发将解决当前蛋白质组学工作中的一个关键空白。

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.