Fully Automatable Multidimensional Reversed-Phase Liquid Chromatography with Online Tandem Mass Spectrometry

: Liquid chromatography (LC) is essential for sample fractionation in shotgun proteomics applications. With suitable design, common LC separation chemistries, including reversed-phase (RP) and strong cation exchange (SCX) mode, can be combined in online multidimensional LC to greatly enhance the overall separation power and, thus, proteome coverage. This protocol describes the design and assembly of a flexible online multidimensional RP-SCX-RP LC system that is compatible with deep proteome profiling on common shotgun proteomics platforms.