ISOLATION OF HYDROPHOBIC LIPOPROTEINS IN ORGANIC-SOLVENTS BY PRESSURE-ASSISTED CAPILLARY ELECTROPHORESIS FOR SUBSEQUENT MASS-SPECTROMETRIC CHARACTERIZATION

Two capillary electrophoretic (CE) separation techniques with either s imultaneous solvent flow induced by hydrostatic pressure or CE followe d by low pressurization with helium were developed for the analysis of extremely hydrophobic proteins, such as the lung surfactant protein S P-C. For both related procedures, buffer solutions containing up to 70 % of 2-propanol were used for the capillary electrophoretic separation . This high concentration of organic co-solvent, needed to solubilize the protein, dramatically reduces the electroosmotic flow (EOF) in ami nopropyltrimethoxysilane-treated fused-silica capillaries. Because the EOF was insufficient to elute the separated analytes from the capilla ry, two ''pressure-assisted'' CE techniques were developed. An additio nal flow to elute the separated analytes was produced either by raisin g the inlet of the capillary or by helium pressure. Using the pressuri zation procedure a baseline separation of the SP-C protein and its dim eric complex was obtained in a 55-minute electrophoretic run, followed by pressure elution of the analyte to the detector. The present combi nation of pressurization and capillary electrophoresis does not requir e any detergents or involatile buffer additives, which are usually nee ded to solubilize extremely hydrophobic lipoproteins. It is therefore applicable to on-line coupling with electrospray mass spectrometry for the direct structural characterization of hydrophobic proteins.