Development of multi-dimensional separation schemes for the analysis of membrane proteins.

Abstract

The heterogeneity of cellular protein expression has stimulated development of separations targeting smaller groups of related proteins rather than entire proteomes. The following research describes the development of multidimensional liquid phase techniques for the characterization of membrane subproteomes from human breast epithelial cell lines. Embedded in or associated with the cell's lipid bilayer, membrane proteins are typically excluded from, or vastly underrepresented in, most analyses of whole cell lysates. Creating an enriched membrane protein fractionate enhances detection of lower abundance membrane proteins and allows separations to be tailored around their unique properties. Evaluation of several traditional techniques was performed to assess the difficulties incurred during membrane protein separations. Maintenance of adequate membrane protein solubility using the most current detergent and chaotrope mixtures during both native and denaturing electrophoretic separations (NPAGE, LIEF, and IPG-IEF) was unsuccessful. Separation of membrane proteins using chromatographic methods was extensively investigated. The comparison of human MCF 10 breast epithelial membrane extracts was achieved through optimized separations using NPS-RP-HPLC coupled with 1D-SDS-PAGE with protein identification by MALDI-TOF-MS and MALDI-QTOF-MS/MS. Subsequently, a 2D-LC membrane protein expression map was generated by coupling chromatofocusing with NPS-RP-HPLC. The 2D-LC map provides a convenient way to view membrane protein expression as a function of protein isoelectric point and hydrophobicity. Liquid phase separations facilitate the automated isolation and collection of protein bands/peaks of interest. Development of intact protein separations is essential for identification of novel proteins and protein biomarkers that can be collected for more targeted studies. Such approaches support potential further characterization and sequencing of novel and unknown proteins, which is vital for developing and annotating current protein and peptide databases in order that they may contain accurate mass tags for every DNA-encoded protein isoform. Identification and characterization of cell surface protein biomarkers in human cancer cell lines using the developed liquid phase methods can lead to the development of diagnostic and prognostic indicators of disease as well as therapeutic agents.