A fully automated dual-online multifunctional ultrahigh pressure liquid chromatography (DO-MULTI-UPLC) system has been developed for high throughput proteome analyses of complex peptide mixtures. The system employs two online solid phase extraction (SPE) columns (150μm inner diameter×3cm), two capillary reverse phase (RP) columns (75μm×100cm) and a strong cation exchange (SCX) column (150μm×15cm) on a single system utilizing one binary pump and one isocratic pump. With the automated operation of six switching valves, the selection of LC experiments between single-dimensional RPLC and online two-dimensional SCX/RPLC were achieved automatically, without manual intervention, while two RPLC columns were used independently and alternatively. By essentially removing the dead time for column equilibration between experiments, in either 1D mode or 2D experimental mode, the current system was demonstrated to increase the experimental throughput by about two folds, while keeping the inter-column reproducibility of peptide elution time in less than 1% of gradient time. The advantageous features of the proposed system were demonstrated by its application to proteome samples of varying complexities.
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