Separation Methods in Membrane Proteomics -Integration of Aqueous Two-Phase Partitioning for Enrichment and Pre-Fractionation

Abstract: The study of the protein complement of a genome is called proteomics. Proteomics can be defined as identification, quantification and characterization of a total set of proteins expressed at different time-points or under different cellular states to provide global information about biological processes. The study of membrane proteins is a challenging area in the development of proteomic research. Despite their importance in biology and medicine, membrane proteins are remarkably underrepresented in the majority of current proteomic studies, which is largely due to poor solubility in aqueous solutions and thus incompatibility with the traditional separation method, two-dimensional gel electrophoresis. This stresses the need for finding efficient strategies for enrichment and pre-fractionation, and development of novel high-resolution separation techniques for progress of membrane proteomics. In this thesis, aqueous two-phase partitioning has been studied for enrichment and pre-fractionation of proteins to facilitate membrane proteomes analysis. Aqueous two-phase partitioning is a method used in different bioseparation applications for initial recovery of biomolecules mainly based on their surface properties. A number of different aqueous two-phase systems have been used and evaluated with a wide variety of proteomic separation and identification techniques. Detergent/polymer aqueous two-phase partitioning was shown to be efficient for pre-fractionation of yeast mitochondrial proteins. Furthermore, the structural integrity of multi-subunit membrane protein complexes was maintained in the detergent/polymer two-phase partitioning experiments. The importance of screening experimental conditions for optimized extraction of membrane proteins was demonstrated by the efficient and non-denaturing solubilization obtained by the combination of zwitterionic and non-ionic detergents. Metal affinity two-phase partitioning was investigated for selective enrichment of inner membrane vesicles from E. coli and provided a rapid enrichment compared to traditional sucrose gradient centrifugation. PEG/dextran two-phase partitioning was employed for rapid and efficient enrichment of plasma membranes from human peripheral blood mononuclear cells. The identification of a number of unique signature peptides derived from nine cell surface-specific CD-antigens showed that the proposed strategy can potentially be used for proteomic monitoring of disease reactions and therapeutic treatment on membrane bound cell surface proteins. In conclusion, reduction of sample complexity is a key issue in all proteomic studies but especially so in membrane proteomics. The use of aqueous two-phase partitioning for enrichment and pre-fractionation combined with efficient high-resolution separation techniques will provide a useful approach for membrane proteins and membrane protein complexes in future proteomic studies.