Automated signature peptide approach for proteomics

This paper addresses the issue of automating the multidimensional chromatographic, signature peptide approach to proteomics. Peptides were automatically reduced and alkylated in the autosampler of the instrument. Trypsin digestion of all proteins in the sample was then executed on an immobilized enzyme column and the digest directly transferred to an affinity chromatography column. Although a wide variety of affinity columns may be used, the specific column used in this case was a Ga(III) loaded immobilized metal affinity chromatography (IMAC) column. Ga(III)-IMAC is known to select phosphorylated peptides. Phosphorylated peptides selected by the affinity column from tryptic digests of milk were automatically transferred to a reversed-phase liquid chromatography (RPLC) column. Further fractionation of tryptic peptides on the RPLC column was achieved with linear solvent gradient elution. Effluent from the RPLC column was electrosprayed into a time-of-flight mass spectrometer. The entire process was controlled by software in the liquid chromatograph. With slight modification, it is possible to add multiple columns in parallel at any of the single column positions to further increase throughput. Total analysis time in the tandem column mode of operation was under 2 h.