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 electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress 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 protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins.
Barnidge
D R
DR
Department of Chemistry and Biochemistry, Montana State University, 108 Gaines Hall, Bozeman, Montana 58717-3520, USA.
Dratz
E A
EA
Jesaitis
A J
AJ
Sunner
J
J
D001436:Bacteriorhodopsins / Q000032:analysis* / Q000737:chemistry; D002457:Cell Extracts / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D019617:Halobacterium salinarum / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D012995:Solubility; D012996:Solutions
D002457:Cell Extracts; D003902:Detergents; D008565:Membrane Proteins; D010455:Peptides; D012996:Solutions; D001436:Bacteriorhodopsins