Selective Enrichment of Membrane Proteins by Partition Phase Separation for Proteomic Studies.

M Walid Qoronfleh,Ray Ignacio,Barbara Kaboord,Betsy Benton

doi:10.1155/s1110724303209244

Abstract

The human proteome project will demand faster, easier, and more reliable methods to isolate and purify protein targets. Membrane proteins are the most valuable group of proteins since they are the target for 70–80% of all drugs. Perbio Science has developed a protocol for the quick, easy, and reproducible isolation of integral membrane proteins from eukaryotic cells. This procedure utilizes a proprietary formulation to facilitate cell membrane disruption in a mild, nondenaturing environment and efficiently solubilizes membrane proteins. The technique utilizes a two-phase partitioning system that enables the class separation of hydrophobic and hydrophilic proteins. A variety of protein markers were used to investigate the partitioning efficiency of the membrane protein extraction reagents (Mem-PER) (Mem-PER is a registered trademark of Pierce Biotechnology, Inc) system. These included membrane proteins with one or more transmembrane spanning domains as well as peripheral and cytosolic proteins. Based on densitometry analyses of our Western blots, we obtained excellent solubilization of membrane proteins with less than 10% contamination of the hydrophobic fraction with hydrophilic proteins. Compared to other methodologies for membrane protein solubilization that use time-consuming protocols or expensive and cumbersome instrumentation, the Mem-PER reagents system for eukaryotic membrane protein extraction offers an easy, efficient, and reproducible method to isolate membrane proteins from mammalian and yeast cells.

Highlights

Based on the sequences from several genomes, transmembrane proteins have been predicted to comprise approximately 30% of eukaryotic proteomes [1]
We show that hydrophilic proteins are recovered in the aqueous phase whereas integral membrane proteins are enriched in the detergent phase
The membrane protein extraction protocol was accomplished in two parts (Figure 1)

Summary

Introduction

Based on the sequences from several genomes, transmembrane proteins have been predicted to comprise approximately 30% of eukaryotic proteomes [1]. Membrane proteins are the most elusive and the most sought after proteins in drug discovery They play a key role in signal transduction, cell adhesion, and ion transport and are important pharmacological targets. Because of their hydrophobic and basic nature, and frequently large size, their isolation is not easy. Techniques used for membrane protein isolation include gradient separation [2], polymer partitioning [3], and chemical treatment [4]. These methods typically result in high purity but low recovery and, with the exception of polymer partitioning, are time consuming. Detergent extraction combined with ultracentrifugation is by far the most commonly used method for membrane protein isolation [5, 6, 7]; this method is a multistep process involving mechanical disruption of cells followed by lengthy centrifugation prior to solubilization of the proteins in detergent

Methods

Results

Conclusion