Rapid and effective focusing in a carrier ampholyte solution isoelectric focusing system: a proteome prefractionation tool.
Preparative (solution) isoelectric focusing (sIEF) is a proven technique for proteome prefractionation, but carries limitations which include the risk of protein loss from isoelectric precipitation, poor focusing, and excessively long separation times. This report describes a simple yet effective method to achieve rapid focusing (as fast as 1 h) and maximize protein recovery using a carrier ampholyte sIEF system. Cathodic drift was not present over the time course of the experiment using our eight-chamber device, and we demonstrate the effectiveness of this device for focusing proteome mixtures. We also discuss an MS-compatible acidic wash protocol, which is shown to enhance the recovery of proteins following sIEF, thus, improving detection by LC-MS/MS. These approaches overcome significant shortcomings of the technique, enabling effective prefractionation prior to MS analysis.
Tran
John C
JC
Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Doucette
Alan A
AA
D000665:Ampholyte Mixtures / Q000737:chemistry*; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods
D000665:Ampholyte Mixtures; D011506:Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins