Development of an online peptide fractionation system comprising a multiphasic LC chip that integrates reversed phase and strong cation exchange chromatography upstream of the MS based on the multi-dimensional protein identification technology (mudPIT). Chips were assessed in terms of chromatographic as well as protein and peptide identification reproducibility using five-step fractionation of 0, 2, 50, 500 and 1500 mM ammonium acetate. Further, a comparison of multiphase LC chip protein and peptide identification versus conventional reversed phase LC-MSMS was performed.