On-line, Chip-Based Strategy for Increased Depth of Coverage in Proteomic Samples

On-line two-dimensional (2D) liquid chromatography is widely used for protein identification and quantification because of the advantage of increased peak capacity. A common workflow is the combination of strong cation exchange (SCX) as an orthogonal first dimension to reversed-phase (RP) chromatography as the second dimension. A more recently developed strategy has emerged where the first dimension is a high pH RP separation which has less orthogonality, but provides higher peak capacity compared to SCX. These 2D workflows are essential for measuring lower abundant proteins in complex proteomes, however can be complex to implement. In this application note, a simplified chip based 2D-LCMS workflow using a high pH/RP first dimensional separation and a low pH/RP secondary dimension coupled directly to mass spectrometer for proteomic analysis is described.