A novel and large-scale proteomic method has been established and applied for profiling mitochondria proteome of human fetal liver. The experimental results showed that combination of different orthogonal separation methods followed by capillary reversed-phase liquid chromatography and on-line electrospray ionization tandem mass spectrometry expanded the dynamic range and resolution, and enabled the identification of 915 proteins from 2977 different peptides. After deletion of redundant proteins among different batches of data, 477 proteins were identified, in which 291 proteins were unique proteins and 186 proteins were cluster proteins. 144 proteins were clearly localized in mitochondria of human fetal liver. The molecular weights of the identified proteins ranged from 7000 Da to 330000 Da and pI values from 4.0 to 11.89. These results demonstrated that liquid phase based separation could overcome some shortcomings adherent to two-dimensional gel-electrophoesis. However, some common issues, such as protein clusters or protein groups and peptide number limited for a reliable protein identification existed in both gel based separation and liquid phase based separation, still have to be addressed in the future researches.
