Displacement chromatography, an elution mode already introduced more than 70 years ago, has been described to offer some advantages over gradient chromatography like high separation efficiency, no need for salts in ion exchange chromatography and enrichment of low abundant molecules. Recently our group demonstrated that displacement chromatography is applicable and beneficial also for bottom up proteomics of complex protein mixtures (Trusch et al. 2012). However, in that online two-dimensional liquid chromatography mass spectrometry (2D-LC-MS) approach the total run time was unacceptable long. In a new study a much smaller cation exchange column (CEC) was chosen with a bed volume of 120 nl and a total binding capacity of approximately 5 µg tryptic peptides. The CEC was integrated into a LC-MS system. The performance of this 2D-LC-MS system for identifying the proteins in a complex tryptic peptide mixture (HeLa protein digest standard) was compared, using either the displacement (DM) or the gradient mode (GM) in the first dimension. With DM a significantly better separation efficiency was achieved, especially for peptides which are doubly positive charged in the eluent at a pH of 2.3 and are the majority of tryptic peptides in mammalian proteomes. The better separation in DM is responsible for a better reproducibility, larger number of identified peptides (> 2.5-fold increase for doubly charged peptides) and proteins including higher protein sequence coverages. In conclusion, the study demonstrates that the displacement mode for CEC in the first dimension of 2D-LC-MS is clearly better than the gradient mode and can already be used for total peptide amounts less than 5 µg.