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 simultaneous solvent flow induced by hydrostatic pressure or CE followed by low pressurization with helium were developed for the analysis of extremely hydrophobic proteins, such as the lung surfactant protein SP-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 aminopropyltrimethoxysilane-treated fused-silica capillaries. Because the EOF was insufficient to elute the separated analytes from the capillary, two "pressure-assisted" CE techniques were developed. An additional flow to elute the separated analytes was produced either by raising the inlet of the capillary or by helium pressure. Using the pressurization procedure a baseline separation of the SP-C protein and its dimeric complex was obtained in a 55-minute electrophoretic run, followed by pressure elution of the analyte to the detector. The present combination of pressurization and capillary electrophoresis does not require any detergents or involatile buffer additives, which are usually needed 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.
Weinmann
W
W
Fakultät für Chemie, Universität Konstanz, Germany.
Maier
C
C
Baumeister
K
K
Przybylski
M
M
Parker
C E
CE
Tomer
K B
KB
D000595:Amino Acid Sequence; D004586:Electrophoresis / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry / Q000302:isolation & purification*; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012997:Solvents
D010169:Palmitic Acids; D011510:Proteolipids; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins; D012997:Solvents; D019308:Palmitic Acid