Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry

The analysis of integral membrane proteins or transmembrane peptides by ele ctrospray ionization mass spectrometry (ESI-MS) is difficult since detergen ts, used to solubilize these hydrophobic proteins and peptides, severely su ppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids pr otein precipitation and resolubilization. Detergent-solubilized bacteriorho dopsin is extracted into a nonpolar solvent phase by adding a chloroform/me thanol/water solvent mixture to the aqueous detergent solution. ESI mass sp ectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequ ences were seen in all mass spectra. Additional peaks were likely due to in tact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic gro up attached, and to bacterioopsin associated with lipid molecules. A separa tion process that occurred in the fused-silica capillary leading to the ele ctrospray tip was essential for obtaining ESI mass spectra of bacterioopsin . The extraction-into-chloroform procedure also worked well with hydrophobi c, transmembrane-type peptides that were insoluble in other electrospray so lvents, including 100% formic acid, and the method has application to trans membrane peptides formed from digests of integral membrane proteins. (C) 19 99 Academic Press.