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Determination of the proteome and identification of biomarkers is required to monitor dynamic changes in living organisms and predict the onset of an illness. One popular method to tackle contemporary proteomic samples is called shotgun proteomics, in which proteins are digested, the resulting peptides are separated by high-performance liquid chromatography (HPLC), and identification is performed with tandem mass-spectrometry. Digestion of proteins typically leads to a very large number of peptides. For example digestion of a cell lysate easily generates 500,000 peptides. The separation of these highly complex peptide samples is one of the major challenges in analytical chemistry. The main strategy to improve the efficiency of packed columns is either to increase column length or by decreasing the size of the stationary phase particles. However, to operate these columns effectively the LC conditions need to be adjusted accordingly. Naturally, the on-line coupling to MS systems has to be taken into account in the optimization process. Here, we report on the performance of nanoLC columns operating at ultra-high pressure. The effects of column parameters (particle size and column length) and LC conditions (gradient time, flow rate, column temperature) were investigated with reversed-phase (RP) gradient nanoLC. High-resolution LC-MS separations of complex proteomic peptide samples are demonstrated by combining long columns with 2 μm particles and long gradients. The effects of LC parameters on performance and the influence on peptide identification are discussed.