Application Of AACT/SILAC In Proteomics Analysis Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is known as a one of difficultly curable diseases in the world and in China. Surgery remains the primary treatment of choice, but only approximately 6% of patients survive more than five years after resection. The major obstacle for the prolonging survival of HCC patients is metastasis after resection. Meanwhile the molecular mechanism underlying the HCC still has yet to be completely understood. Efforts to assess proteins involved in HCC are critical to elucidate potential biomarkers and therapy targets in HCC. Quantitative proteomics or comparative proteomics aims to screen the proteome differences associated with disease phenotype, and discovers the proteins functioning in the process of HCC. Systematic understanding using quantitative proteomics by comparing the HCC cell line versus liver cell line or by comparing HCC cell lines with different metastasis potential will show light on the molecular mechanisms of HCC and provide potential biomarkers for early diagnosis and therapy.The present works developed one method to determine the false positive of labeled peptide in amino acid-coded mass tagging/stable isotope labeling with amino acids in cultured cell (AACT/SILAC). The works also comprehensively profiled the differential protein expression pattern using AACT/SILAC in HCC, and provided many useful biomarker candidates for HCC.Firstly the work provided a brief summary about the development of quantitative proteomics, introducing the MS instrument and quantification methods such as 2DE,ICAT,iTRAQ and AACT/SILAC.Secondly the work improved the AACT/SILAC technology and focused on the differential proteome of HCC, and was illustrated as following.(1) Evaluation of the false positive of labeled peptide in AACT/SILAC Stable isotope labeling became the chief technology in quantitative proteomics and false positive of peptide labeled was not well studied yet for the present. This article developed a method to judge the false positive of labeled peptide. Based on the probability model of isotope distribution, theoretical isotope distribution with different label efficiency were calculated, the label efficiency of peptide labeled was evaluated by comparing the experimental and theoretical isotope distribution using the least-square method. The peptide was determined as a false-positive one when its label efficiency was below the threshold. This judgment method would facilitate the precisely quantification of AACT/SILAC in proteomics.(2) Proteome expression profile of HCC cell line with high metastasis potentialProteome expression profile was the basis to understand the HCC mechanism and therapy. Proteins identified in HCC cell lines with high metastasis potential were put into biological context with various annotation sources followed by a bioinformatics analysis. The profile was annotated in genomic context and characterized the correlation between subcellular location and the pI. Proteins were further annotated with GeneOntology, grouped into proteins families in InterPro database and mapped to KEGG pathways they were involved. Advantages of AACT/SILAC over 2DE in identifying the proteins with high molecular weight and pI or membrane proteins were demonstrated. The protein-protein interaction network derived from HPRD was also examined.(3) Differential proteome profile between a HCC cell line and liver cell lineProteome alteration between two cell lines derived from HCC tissue and its adjacent tissue were screened using AACT/SILAC and differential proteome profile containing 224 proteins were obtained. Western blotting and immune histochemistry also validated the accuracy of the quantification results and the expression alteration of representative proteins in clinical HCC tissues. Biological functions of differentially expressed proteins were annotated by GeneOntology, KEGG and protein-protein interaction. These proteins were mapped to MAPK, calcium, Wnt signal pathway which regulated the cell proliferation and cell migration and functioned in HCC. Functions of annexin family, heat shock proteins and cytokeratin family in HCC were also disscused.(4) Differential proteome profile between HCC cell lines with different metastasis potentialProteome profile of two HCC cell lines with high and low metastasis potential, respectively, were compared using AACT/SILAC coupled with SDS-PAGE-LC-MALDI -MS and a nonredundant dataset of 360 proteins were quantified as metastasis-related proteins. Expression differences were further validated by Western blotting in a series of HCC cell types with progressively increasing trend of metastasis and in clinical samples. Function annotations were also analyzed by GeneOntology, KEGG and protein-protein interactions, illustrating their biological roles in cell proliferation, apoptosis, cell migration and tumor angiogenesis.Overall, proteome alteration in HCC and metastasis in HCC were systematically screened using AACT/SILAC, and a nonredundant protein dataset involved in HCC was identified here. The dataset contained a set of proteins related with HCC development, facilitating our understanding on the molecular mechanism of HCC and metastasis, and would benefit the early diagnosis and clinical treatment in HCC patients.