The Aact Technology For Qsg-7701/qgy-7703 Quantitative Proteome Study And Search For Related Molecular Markers Of Liver Cancer,

The thesis was focused on quantitative proteomics platform of mass spectrometry based amino acid-coded tagging(AACT) technique.To investigate candidates of diagnostic or therapeutic biomarkers for liver cancers,we comprehensively measured systematic changes in protein expression of human hepatocellular carcinoma(HCC). The effective treatment of liver cancer relies on the diagnosis of the disease at an early stage.The research work of this dissertation was composed of four parts.The first parts was about A novel method of quaternizing amino acid/peptide and its product in situ analysis using ESI-MS;In the second part,we set up a high throughput platform for global quantitative exploration of proteome using AACT proteomics technologies; The third part was comparative proteomic analysis of two paired HCC cell lines with the same genetic background to investigate markers for HCC and metastasis.And the last part was optimization routine technology for organelles.The details were described as the chapters in the following:(1) The preface part summarized recent research progresses on quantitative proteomics and in the context of their respective strengths/weakness,especially emphasized the application of major amino acid-coded mass tagging based quantitative proteomics approaches.(2) A new method of quaternizing amino acid and peptide was reported.This method could offer a potential chance to combine quanternizing and mass spectrometry which is helpful for the future use of quanternizing reaction in quantitative proteomics.(3) We set up a high throughput platform for global exploration of quantitative proteome based on the amino acid coded-mass tagging technologies.Content includes cell labeling,choice of isotope amino acid,the label degree detection,LC-MS separates the optimization of parameter's,threshold value setting for quantification, quantify standard build up et.al..Finally we set up a platform for the following work in quantification and protein-protein interaction.(4) Comparative proteomic analysis of a paired human liver normal-carcinoma cell lines with the same genetic background to investigate hepatocellular carcinoma Markers.Quantitative proteomic analysis of two cell lines derived from healthy liver and carcinoma tissue of a same donor,i.e.,QSG-7701 for the normal and QGY-7703 for the cancer cells respectively,was conducted using AACT.Among a total of 1343 proteins,511 was precisely quantified in HCC cells.According to their previously characterized functions,the differentially expressed proteins were found associated with five major functional categories.The accuracy of the AACT-based quantification was validated by Western blotting.Furthermore,the differentially expressed proteins were identified in HCC specimen.The consistency between cell lines and clinic tissues,comprised a part of reported biomarker for HCC.Altogether our study provides some insights into the molecular events that may lead to diverse strategies for diagnosis and treatment of HCC.(5) Comprehensive profiling of metastasis-related proteins in paired hepatocelluar carcinoma cells with different metastasis potential.Metastasis is the primary cause of death in hepatocelluar carcinoma(HCC) patients.To identify those proteins associated with metastasis potential,we have conducted comparative proteomic analysis on a pair of the HCC cell lines characterized with different metastasis potential originated from a same donor(MHCC97L versus MHCC97H) by employing an amino acid-coded mass tagging(AACT)-based quantitative proteomic method.(6) We optimized routine technology for organelle,especially for nucleus and mitochondrial in three level cells,mice tissues and healthy human liver.