A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis.
Two-dimensional electrophoresis is a critical technique for proteome research, but currently available methods are not capable of resolving the >10,000 protein components in most eukaryotic proteomes. We have developed and demonstrated the utility of a novel solution isoelectrofocusing device and method that can reproducibly prefractionate cell extracts into well-defined pools prior to 2D PAGE on a scale directly compatible with the high sensitivity of proteome studies. A prototype device was used to separate metabolically radiolabeled Escherichia coli extracts in method optimization and proof-of-principle experiments. Samples were loaded into separation chambers divided by thin polyacrylamide gels containing immobilines at specific pH values and isoelectrically focused for several hours, which resulted in well-resolved fractions. Total recoveries in the fractionated samples were greater than 80% and most protein spots in the original sample were recovered after this prefractionation step. Nonideal behavior (precipitation/aggregation), typically encountered when unfractionated samples at high protein loads were applied directly to either narrow- or broad-range IPG gels, was dramatically reduced. Hence this approach allows increases in overall protein loads, resolution, and dynamic detection range compared with either alternative prefractionation methods or direct use of parallel narrow pH range gels without sample prefractionation. The pH ranges and number of fractions can be readily adapted to the requirements of specific types of samples and projects. This method should allow quantitative comparisons of at least 10,000 protein components on a series of narrow pH range gels, and protein detection limits are estimated to be 1000 molecules per cell when mammalian proteomes are fractionated into five or more pools.
Zuo
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The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania, 19104, USA.
Speicher
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D002457:Cell Extracts / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D020543:Proteome / Q000737:chemistry*; D012996:Solutions
D002457:Cell Extracts; D020543:Proteome; D012996:Solutions