On-line parallel reversed phase two-dimensional liquid chromatography for high-throughput analysis of complex proteomic samples

A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is constructed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm x 4.6 mm I.D.) followed by reversed phase chromatography (Hypersil BIDS C18, 15 mm x 4.6 mm I.D.) to separate the complex peptides from globin peptic hydrolysate. After the sample was loaded on the SCX column, the phosphate buffer (pH 4.0) was used to elute the peptides. Then, elutes flowed through the interface and the peptides focused on the head of the trapping columns (Hypersil BIDS C18, 15 mm x 4.6 mm I.D.) but salt passed into the waste. After the valve was switched, the samples were flushed with a backward flow into the RP analytical column. The peptides on the SCX were eluted With 12 discontinuous steps linearly increasing salt concentrations. The peptides enriched on the trapping column were desalted and separated by the RIP columns. The resolution and the resolved peaks of the 2D SCX/RP system were greatly increased and the total peak capacity reached as high as 2280