Coacervates for Extraction and Enrichment of Membrane Proteins from Biological Matrices in Proteomics

北卡罗来纳州立大学的Morteza G.Khaledi教授和David C.Muddiman教授得到了化学系化学测量和成像(CMI)计划的支持，他们研究了新发现的被称为全氟醇诱导凝聚(PFAIC)的超分子液体用于加强从生物基质中选择性提取和浓缩膜蛋白。膜蛋白是细胞表面膜最重要的组成成分。它们扮演着高度选择性的细胞受体和守门人的角色，调节关键营养物质和能量的进出细胞。将生物样品中的膜蛋白溶解、提取和浓缩到适合进一步分析的水溶液中--这是表征单个蛋白质和了解其细胞功能的关键步骤--是当代生物分析化学和分子生物学中的一个相当大的挑战领域。该项目有望解决这一挑战，并为膜蛋白的鉴定提供新的途径，从而促进我们对细胞功能和生命过程的理解。在进行该项目的过程中，研究生和本科生将在生物分析分离技术、蛋白质组学和质谱学方面获得宝贵的技能。通过与北卡罗来纳州立大学凯南研究所的凯南研究员计划的合作，还制定了计划，让高中教师参与与这项研究相关的一般领域的暑期研究和高中课程材料的开发。拟议研究的总体目标是探索使用全氟醇诱导凝聚(PFAIC)来加强从生物基质中选择性提取和浓缩膜蛋白。这项研究分为四个具体目标：(1)通过对不同结构和物理化学性质的蛋白质以及不同化学组成的PFAIC系统的分配模式、萃取效率和浓缩因子的系统研究，了解PFAIC系统中蛋白质的提取原理；(2)研究膜蛋白在PFAIC相中的浓缩一般策略；(3)以酵母为模型生物样品，研究PFAIC在蛋白质组学分析中用于膜蛋白的预分离、提取和浓缩的有效性；(4)探讨诱导细胞膜脂质双层凝聚和膜结合蛋白增溶的可能性。

Professors Morteza G. Khaledi and David C. Muddiman of North Carolina State University are supported by the Chemical Measurement and Imaging (CMI) Program in the Division of Chemistry to investigate the use of newly discovered supramolecular liquids called Perfluoro Alcohol Induced Coacervates (PFAIC) for enhanced selective extraction and enrichment of membrane proteins from biological matrices. Membrane proteins are the most important cell surface membrane constituents. They act as highly-selective cellular receptors and gatekeepers, regulating the traffic of critical nutrients and energy in and out of the cells. Solubilization, extraction, and enrichment of membrane proteins from biological samples into aqueous solutions suitable for further analysis - a critical step for the characterization of the individual proteins and for understanding their cellular function - is an area of considerable challenge in contemporary bioanalytical chemistry and molecular biology. The project promises to tackle this challenge and provide new avenues for the identification of membrane proteins and hence advance our understanding of cellular function and life processes. In the course of conducting the project, graduate and undergraduate students will gain valuable skills in bioanalytical separation technologies, proteomics and mass spectrometry. Plans are also established, through a partnership with the Kenan Fellows Program of the Kenan Institute at North Carolina State University, to involve high school teachers in summer research and in the development of high school curriculum materials in the general areas related to this research.The overarching objective of the proposed research is to explore the use of Perfluoro Alcohol Induced Coacervates (PFAIC) for enhanced selective extraction and enrichment of membrane proteins from biological matrices. The proposed research is organized into four specific aims: (1) to achieve an understanding of the principles of extraction of proteins in PFAIC systems through systematic studies of partitioning patterns, extraction efficiency and enrichment factors of proteins with different structural and physico-chemical properties and PFAIC systems with different chemical composition; (2) to investigate general strategies for enrichment of membrane proteins into PFAIC phases; (3) to investigate the effectiveness of PFAIC for pre-fractionation, extraction and enrichment of membrane proteins in shot-gun bottom-up proteomics analysis of yeast as a model biological sample; and (4) to explore the possibility of inducing coacervation of lipid bilayers in cell membranes and concomitant solubilization of the membrane bound proteins.