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

北卡罗莱纳州立大学的Morteza G. Khaledi教授和David C. Muddiman教授在化学系化学测量和成像（CMI）项目的支持下，研究了新发现的称为全氟醇诱导凝聚物（pfac）的超分子液体的使用，以增强从生物基质中选择性提取和富集膜蛋白的能力。膜蛋白是细胞表面最重要的膜成分。它们作为高度选择性的细胞受体和看门人，调节关键营养物质和能量进出细胞的交通。在当代生物分析化学和分子生物学中，将生物样品中的膜蛋白溶解、提取和富集到适合进一步分析的水溶液中是表征单个蛋白质和了解其细胞功能的关键步骤，也是一个相当具有挑战性的领域。该项目有望解决这一挑战，并为鉴定膜蛋白提供新的途径，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，研究生和本科生将获得生物分析分离技术，蛋白质组学和质谱学的宝贵技能。通过与北卡罗莱纳州立大学凯南研究所的凯南研究员方案建立伙伴关系，还制定了计划，使高中教师参与与这项研究有关的一般领域的夏季研究和高中课程材料的编制。所提出的研究的总体目标是探索使用全氟醇诱导凝聚体（pfac）从生物基质中增强选择性提取和富集膜蛋白。本研究主要分为四个方面：(1)通过系统研究具有不同结构和理化性质的蛋白质和具有不同化学成分的pfac系统的分配模式、提取效率和富集因子，了解pfac系统中蛋白质的提取原理；(2)研究膜蛋白富集到pfac相的一般策略；(3)以酵母为模型生物样品，研究pfac对膜蛋白的预分离、提取和富集的有效性；(4)探索诱导细胞膜脂质双分子层的聚集和伴随的膜结合蛋白的增溶的可能性。

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.