Two-dimensional Difference Chromatography Combined With Isotope Affinity Tags - Identified By Mass Spectrometry For Relative Quantification Of Proteome Research And Applications

After the accomplishment of the Human Genome Project, the emphasis of the life science research has shifted from the structural genomics to the functional genomics. As one of the most important areas of functional genomics, proteomics mainly focuses on studying the genes' products and their functions. Recently, on the basis of the mature protein identification, a lot of researchers have been more interested in quantitative proteomics because it can help know the function of proteins, the network of the protein-protein interaction, the target of drug discovery and the protein markers for diagnosis.In the present, the method for quantitative proteomics can be divided into two kinds. One is the method that combines two dimensional electrophoresis with the image analysis, the other is that that combines the stable isotopic label with the mass spectrometry analysis. Among the various methods of the latter, the isotope affinity coded tag (ICAT) is the most widely used method for its high accuracy, the capacity to be directly applied to the sample in vitro and the ability to decrease the complexity of the sample. For the facility of the combination of the LC and ESI-MS, a lot of researchers use the method to perform ICAT quantitative proteomics, but its detection sensitivity is low, and MS analysis suffers from the on-line detection time of LC separation. In this experiment, we used automatic Probot system to combine the LC and MALDI-TOF-TOF MS. The advantage of the method is convenient time arrangement for the analysis by MALDI-TOF-TOF MS, the result dependent MSMS acquisition, and making full use of the high sensitivity and high speed of MALDI-TOF-TOF MS which results in the increased analysis effitiency and short analysis time. First, we used standard proteins to establish the quantitative proteomics method based on cICAT and LC-MALDI-TOF-TOF MS, and then tested the accuracy, reproducibility and dynamic range of the method. The result shows that the strategy has good accuracy and reproducibility, and can be applied to the accurate relative quantitation of proteome even in the dynamic range of 30 fold. At the end, the method has been applied into the research of rat liver regeneration, and identified and quantified 74 differential proteins, among those there are 7 proteins showing 2 fold change.Based on the two methods established successfully in our laboratory including intact protein mixture separation by the two dimensional liquid chromatography, chromatography...