In proteomics study, because there were biases of two-dimensional gel electrophoresis in molecular weights, pH and hydrophobicity, multi-dimensional protein identification technology (MuD-PIT)was proposed. Due to the ...In proteomics study, because there were biases of two-dimensional gel electrophoresis in molecular weights, pH and hydrophobicity, multi-dimensional protein identification technology (MuD-PIT)was proposed. Due to the core part of the technique being digestion of total proteins in the first step, some drawbacks appeared, such as complicating the separation of samples, dominating the influence of proteins with high abundance on the identification of proteins with low abundance and losing some information of proteins,for example, post-translational modification et al. , these resulted in the limitation of applications for the method. Taking into account of those factors mentioned above, when studying the mitochondria proteome of human fetal liver with great significance in life process, Proteome Lab PF2D system (Beckman Coulter, USA) was employed. The fractions of extract from fetal liver tissues by saccharose- density gradient centrifugation were prepared on a high velocity centrifuge. The desalting and solvent exchanging of the fractions with Start buffer (pH8. 5, Beckman Coulter, USA) were carried out on a PD-10(Sephadex G-25, 5 000Da) desalting column (amersham pharmacia biotech, Wikstroms,Sweden) according to the procedures described in its product instruction. Then the treated fractions were separated with chromatofocusing and reversed-phase liquid chromatography in tandem, respectively. After concentration and in-solution digestion of protein fractions,the peptide mixture was lyophilized, then resolved with 5% aqueous acytonitrile plus 0.1% formic acid, and finally analyzed with microLC-ESI-ion trap mass spectrometry(Thermo Finnigan, San Jose, CA, USA) and CapLC-ESI-QTOF mass spectrometry (Waters, USA). Mass spectrometric data was searched against IPI protein database with Thermo SEQUEST (Thermo Finnigan, San Jose, CA, USA) and Mascot. A primary database of mitochondrial proteome of human fetal liver was constructed, which could play central role in the large-scale study of the functions of human liver proteome. By using the technique on profiling mitochondrial proteome of human fetal liver, valuable experimental results were obtained and showed that combination of different orthogonal separation methods followed by capillary reversed-phase liquid chromatography and on-line ESI tandem mass spectrometry could expand the dynamic range and resolution, and enabled the identification of 915 proteins from 2 977 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 about 7 000Da to 330 000Da and pI values from about 4.0-11.89.These results demonstrated that liquid phase based separation could overcome some shortcomings adherent to 2DE. 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.展开更多
文摘In proteomics study, because there were biases of two-dimensional gel electrophoresis in molecular weights, pH and hydrophobicity, multi-dimensional protein identification technology (MuD-PIT)was proposed. Due to the core part of the technique being digestion of total proteins in the first step, some drawbacks appeared, such as complicating the separation of samples, dominating the influence of proteins with high abundance on the identification of proteins with low abundance and losing some information of proteins,for example, post-translational modification et al. , these resulted in the limitation of applications for the method. Taking into account of those factors mentioned above, when studying the mitochondria proteome of human fetal liver with great significance in life process, Proteome Lab PF2D system (Beckman Coulter, USA) was employed. The fractions of extract from fetal liver tissues by saccharose- density gradient centrifugation were prepared on a high velocity centrifuge. The desalting and solvent exchanging of the fractions with Start buffer (pH8. 5, Beckman Coulter, USA) were carried out on a PD-10(Sephadex G-25, 5 000Da) desalting column (amersham pharmacia biotech, Wikstroms,Sweden) according to the procedures described in its product instruction. Then the treated fractions were separated with chromatofocusing and reversed-phase liquid chromatography in tandem, respectively. After concentration and in-solution digestion of protein fractions,the peptide mixture was lyophilized, then resolved with 5% aqueous acytonitrile plus 0.1% formic acid, and finally analyzed with microLC-ESI-ion trap mass spectrometry(Thermo Finnigan, San Jose, CA, USA) and CapLC-ESI-QTOF mass spectrometry (Waters, USA). Mass spectrometric data was searched against IPI protein database with Thermo SEQUEST (Thermo Finnigan, San Jose, CA, USA) and Mascot. A primary database of mitochondrial proteome of human fetal liver was constructed, which could play central role in the large-scale study of the functions of human liver proteome. By using the technique on profiling mitochondrial proteome of human fetal liver, valuable experimental results were obtained and showed that combination of different orthogonal separation methods followed by capillary reversed-phase liquid chromatography and on-line ESI tandem mass spectrometry could expand the dynamic range and resolution, and enabled the identification of 915 proteins from 2 977 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 about 7 000Da to 330 000Da and pI values from about 4.0-11.89.These results demonstrated that liquid phase based separation could overcome some shortcomings adherent to 2DE. 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.
