| Membrane proteins are insoluble in aqueous solutions due to their strong hydrophobic characteristics. The solubility of these proteins is further aggravated by their lipophilic milieu. These proteins play significant roles in the brain. They include the transport of small molecules, cell signaling, proteolysis, and lipid synthesis. Furthermore, membrane proteins such as G-Protein Coupled Receptors and ion-transporters play critical roles in an array of disease conditions and are therefore targets for drug discovery. However, despite their potential significance in biological systems, the structure and function of many membrane proteins, particularly those of low abundance, remain elusive, due to difficulties they pose in isolation and characterization.;Perhaps the most challenging membrane proteins to study are those of the central nervous system (CNS). The proteins are embedded in the high lipid environment of the brain, and as expected, they are highly insoluble in aqueous solutions. Therefore, it is not surprising that many low abundance membrane proteins in the CNS remain unknown.;During the last 30 years, numerous efforts have been made to extract and resolve membrane proteins on 2-Dimensional polyacrylamide gels (2-D gels) for the purpose of identification and characterization, but with minimum success due to poor separation on this system. Here, I describe a robust approach for resolving brain membrane proteins, by first washing the membrane tissue, followed by delipidation, solubilization and monodispersion. 2-D gels show the extracted brain membrane proteins obtained using this approach display a far superior resolution than those extracted from CNS and other membrane tissues prepared by traditional methods.;This method was validated by a series of experimental procedures. First, reconstitution of delipidated proteins with native or synthetic phospholipids abolished the high resolution on 2-D gels. Second, gel filtration resolved the proteins according to their size, thus proving that the proteins were in a monodispersed state while in solution, before they were subjected to 2-D gel electrophoresis. Accordingly, fractionation of the proteins on gel filtration followed by 2-D gel established a 3-Dimensional approach for enriching low abundance membrane proteins. Third, amino acid composition analysis showed a significant abundance of hydrophobic residues in the membrane extract, and fourth, western blot analysis demonstrated the propensity for glycosylation on membrane proteins. Fifth, mass spectrometry (MS) identified membrane proteins sequences with high confidence. Interestingly, while there were identifiable sequences, the majority of protein sequences were identified with zero to little confidence, suggesting they were unidentifiable, possibly due to lack of their existence in MS databases. Finally, the development of a knowledgebase, once implemented into a website, will provide researchers with a virtual library of brain membrane protein sequences identified through this method, which may be resourceful in the study of membrane protein functions.;Since there is neither a validated nor scalable method for extracting, solubilizing, monodispersing, and isolating membrane proteins in enough quantities for identification and characterization, the research presented here may lead to major breakthroughs in discovering novel brain membrane proteins, thereby opening a way to investigating their roles in disease pathways. |
