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 electroext raction, the peptides are trapped on a strong cation-exchange microcartridg e, before analysis by capillary LC-ESI-tandem mass spectrometry, The spectr a obtained by tandem mass spectrometry are searched directly against a prot ein database for identification of the protein from which the peptide origi nated. By minimizing surface exposure of the peptides during electroextract ion, a reduction of the detection limits for protein identification is real ized. The performance of the peptide electroextraction was compared directl y with the standard extraction method for in-gel protein digests, using a s tandard dilution series of phosphorylase B and carbonic anhydrase, separate d by SDS-PAGE. The lowest gel loading in which phosphorylase B was identifi ed using the standard extraction method was 2.5 ng or 25 fmol, and the lowe st gel loading in which phosphorylase B was identified using electroextract ion was 1.25 ng or 12.5 fmol. The design of the microextraction cartridge a llows for direct interfacing with capillary LC, which is crucial for mainta ining low detection limits. Furthermore, this method can be used for high-t hroughput proteomics since it can be easily multiplexed and requires only v oltage control and low pressures (similar to 15 psi) for operation, We beli eve that peptide electroextraction is a significant advance for identificat ion of proteins separated by one-dimensional or two-dimensional gel electro phoresis, as it can be easily automated and requires less protein than conv entional methods.