Peptide electroextraction for direct coupling of in-gel digests with capillary LC-MS/MS for protein identification and sequencing.
An electrophoretic method has been developed for the extraction of peptides following in-gel digests of SDS-PAGE separated proteins. During electroextraction, the peptides are trapped on a strong cation-exchange microcartridge, before analysis by capillary LC--ESI-tandem mass spectrometry. The spectra obtained by tandem mass spectrometry are searched directly against a protein database for identification of the protein from which the peptide originated. By minimizing surface exposure of the peptides during electroextraction, a reduction of the detection limits for protein identification is realized. The performance of the peptide electroextraction was compared directly with the standard extraction method for in-gel protein digests, using a standard dilution series of phosphorylase B and carbonic anhydrase, separated by SDS-PAGE. The lowest gel loading in which phosphorylase B was identified using the standard extraction method was 2.5 ng or 25 fmol, and the lowest gel loading in which phosphorylase B was identified using electroextraction was 1.25 ng or 12.5 fmol. The design of the microextraction cartridge allows for direct interfacing with capillary LC, which is crucial for maintaining low detection limits. Furthermore, this method can be used for high-throughput proteomics since it can be easily multiplexed and requires only voltage control and low pressures (approximately 15 psi) for operation. We believe that peptide electroextraction is a significant advance for identification of proteins separated by one-dimensional or two-dimensional gel electrophoresis, as it can be easily automated and requires less protein than conventional methods.
Timperman
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AT
Department of Chemistry, West Virginia University, Morgantown 26506-6045, USA. atimperm@wvu.edu
Aebersold
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D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification*; D011506:Proteins / Q000032:analysis*; D020539:Sequence Analysis, Protein
D010455:Peptides; D011506:Proteins