A New Method For Quantification Of Acetylation Of Histone H3 In Hepatocarcinoma Cells And Enrichment And Enrichment Of Acetylation

The main contributions of this thesis were as following:first, we quantified the acetylated levels of histone H3in different metastasis potential hepatocellular carcinoma cells using LC separation, methylation isotope labeling, and LTQ-Orbitrap mass spectrometry. This result would exhibit valuable information in the research of hepatocellular carcinoma metastatic; second, we established a method of separation and selective enrichment of acetylated proteins or peptides based on microchip technique combined with MALDI-MS analysis for the first time. The strategy has a great potential application in the discovery of novel acetylated proteins in acetylome studies.Lysine acetylation is a dynamic and reversible posttranslational modification process with important implications in cellular regulation. Researches have revealed the importance of acetylation in many cellular processes including, but not limited to, DNA-protein interactions, transcription activation, protein stability, protein-protein interactions, migration and differentiation. It was also proved that every enzyme involved in glycolysis, gluconeogenesis, urea cycle, tricarboxylic acid cycle, fatty acid metabolism, and glycogen metabolism is acetylated. However, biologically important acetylation sites of low abundance remain undetected because of a large number of background non-acetylated peptides. Combined with the enrichment technique, mass spectrometry was able to provide information on the acetylation sites of proteins and peptides. One promising technique is enrichment through immobilization of anti-acetyllysine antibodies. Histone is a highly conserved protein in cell nucleus. Histone is tending to have a variety of post-translational modifications. It plays an important role in gene expression, transcription and repair. It was reported that the state of the acetylation of histone H3has important clinical significance in prostate cancer, breast cancer, lung cancer and other diseases. However, there are almost no detailed researches about hepatocellular carcinoma.In the first chapter, we give an introduction on the proteomics, protein post-translational modifications, acetylome, and the recent research progress on microfluidic chips, including fabrication techniques, materials and analytical applications. In addition, we review the liver proteomic.In the second chapter, we developed a method of quantitative of histone H3acetylation levels of hepatocellular carcinoma cells. In our experiment, we identified and analyzed the acetylated sites and levels of histone H3in different metastasis potential hepatocellular carcinoma cells using LC separation, methylation isotope labeling and LTQ-Orbitrap mass spectrometry. Six quantitative results of acetylated peptides and four results of methylated peptides were obtained. These observations demonstrate that the levels of acetylated peptides and methylated peptides we identified were greatly decreased with the increased of metastatic potential of hepatocellular carcinoma cells. The results can provide theoretical foundation for development of new drugs in the treatment of hepatocellular carcinoma.In the third chapter, we developed a simple and novel microchip-based antibodies immobilization strategy for identification of acetylated proteins and acetylated sites. The protein A/G beads were captured on the surface of the microchip. The antibodies immobilized via the protein A/G provided high-efficiency specific antigen capture. Acetylated proteins or peptides were enriched by the microchip and detected by SDS-PAGE and MALDI-MS. The results show that the developed method can be used to separate and then enrich acetylated proteins or peptides with high efficiency and selectivity. For analysis of acetylated protein,82.4%recovery of the acetylated protein was attained and more acetylated peptides peaks were observed in the mass spectra after enrichment with the microchip-based technique.