title: Improved method for the analysis of membrane proteins by mass spectrometry.
abstract: Membrane-bound and membrane-associated proteins are difficult to analyze by mass spectrometry, since the association with lipids impedes the isolation and solubilization of the proteins in buffers suitable for mass spectrometry and the efficient generation of positively charged peptide ions by electrospray ionization. Current methods mostly utilize detergents for the isolation of proteins from membranes. In this study, we present an improved detergent-free method for the isolation and mass spectrometric identification of membrane-bound and membrane-associated proteins. We delipidate proteins from the membrane bilayer by chloroform extraction to overcome dissolution and ionization problems during analysis. Comparison of our results to results obtained by direct tryptic digestion of insoluble membrane pellets identifies an increased number of membrane proteins, and a higher quality of the resulting mass spectral data.
authors.affiliation: National Center for Proteomics Research, Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. smirza@mcw.edu
authors.forename: Shama P
authors.initials: SP
authors.lastname: Mirza
authors.forename: Brian D
authors.initials: BD
authors.lastname: Halligan
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Greene
authors.forename: Michael
authors.initials: M
authors.lastname: Olivier
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002725:Chloroform / Q000737:chemistry; D002853:Chromatography, Liquid; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D051381:Rats; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D008565:Membrane Proteins; D002725:Chloroform