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Large‐scale μLC‐MS/MS for silver‐ and Coomassie blue‐stained polyacrylamide gels

2‐DE combined with LC‐MS/MS has become a routine, reliable protein separation and identification technology for proteome analysis. The demand for large‐scale protein identifications after 2‐DE separation requires a sensitive and high‐throughput LC‐MS/MS method. In this report, a simple, splitless, fully automated capillary LC‐MS/MS system was described for the large‐scale identification of proteins from gels stained with either silver or CBB. The gel samples were digested and peptides were extracted using an in‐gel digestion workstation. The peptides were automatically introduced into a capillary column by an autosampler connected to an HPLC pump. A nanoLC pump was then used to deliver the gradient and elute the peptides from the capillary column directly into an LCQ IT mass spectrometer. Neither a peptide trapping setting nor a flow split is needed in this simple setup. The collected MS/MS spectra were then automatically searched by SEQUEST, and filtered and organized by DTASelect. Hundreds of silver‐stained or CBB‐stained Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens proteins separated by denaturing or nondenaturing 2‐DE were digested and routinely analyzed using this fully automated μLC‐MS/MS system. High peptide hits and sequence coverage were achieved for most CBB‐stained gel spots. About 75% of the spots were found to contain multiple proteins. Although silver staining is not commonly thought to be optimal for MS analysis, protein identifications were successfully obtained from silver‐stained 2‐DE spots detected using methods with and without formaldehyde for protein fixation.