Extraction Method for Analysis of Detergent-Solubilized Bacteriorhodopsin and Hydrophobic Peptides by Electrospray Ionization Mass Spectrometry

摘要: The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic and peptides, severely suppress analyte ion formation. This problem has been addressed previously precipitating the protein, removing detergent, resolubilizing protein in a nonpolar solvent. Here, we demonstrate method that avoids precipitation resolubilization. Detergent-solubilized bacteriorhodopsin extracted into solvent phase adding chloroform/methanol/water mixture aqueous detergent solution. ESI spectra nonpolar, chloroform-rich were dominated peaks due bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences seen all spectra. Additional likely intact bacteriorhodopsin, i.e., bacterioopsin retinal prosthetic group attached, associated lipid molecules. A separation process occurred fused-silica capillary leading tip was essential for obtaining extraction-into-chloroform procedure also worked well hydrophobic, transmembrane-type insoluble other solvents, including 100% formic acid, application formed from digests proteins.