Journal
JOURNAL OF PROTEOME RESEARCH
Volume 7, Issue 4, Pages 1761-1766Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr700677u
Keywords
mass spectrometry; prefractionation; preparative electrophoresis; proteomics; solution isoelectric focusing; immobilines; ampholytes; recovery; intact protein; separation
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Preparative (solution) isoelectric focusing (slEF) 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 slEF 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 slEF, thus, improving detection by LC-MS/MS. These approaches overcome significant shortcomings of the technique, enabling effective prefractionation prior to MS analysis.
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