title: Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations.
abstract: Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains.
authors.affiliation: The University of Michigan, Department of Chemistry, Ann Arbor, MI 48109-1055, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhu
authors.forename: David M
authors.initials: DM
authors.lastname: Lubman
mesh_terms: D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011506:Proteins