Quantitative Study On Protein Acetylation

Numerous studies have shown that acetylation is one of the most important post-translational modifications on proteins. HATs and HDACs both regulate levels of acetylation which influences key physiological processes such as chromatin remodeling, gene expression, cellular metabolic regulation, protein stability. Therefore, the aberrant acetylation may affect tumor-suppressor genes leading to the occurrence and development of several diseases, such as cancer, neurodegenerative and cardiovascular disorders. The efficient detection of acetylation on biomarkers in complex biological samples would be useful, including characterization of disease-associated acetylation alterations, identification of new diagnostic biomarkers and therapeutic strategies. Thus it is necessary to develop the method to quantify levels of acetylation effectively in different pathological states.The microarray method is an analysis technique with high throughput, sensitivity and specificity which attracts more and more attention. It is not only a powerful research tool for the proteomics, but also is useful in the clinical application, including early diagnosis, prognosis and treatment evaluation. The microarray provides effective technical support for the development of life sciences.There are two main contributions of this thesis. First, the microarray was developed for quantifying levels of acetylation on targeted protein. This method provides an efficient, quantitative and rapid detection of protein abundance and acetylation on targeted protein in parallel. Meanwhile, this microarray was applied to detect the level of acetylation on vascular endothelial growth factor (VEGF). By quantifying the abundance of protein and acetylation on VEGF in serums from normal and HCC patients, this microarray provides a simply, efficient and quantitative approach to detect acetylation on cancer biomarkers. Secondly, we developed the PUF as the material to immobilize the protein trypsin and improve the efficiency of the protease enzyme.In the first chapter, a review of the proteomics, protein post-translational modifications and recent studies on acetylome is presented. Then we introduced the microarray including its research and application on quantitative detection of protein and post translational modification. The background of cancers and the related research methods are also described. And in the chapter, the aims and significance of this dissertation are introduced.In the second chapter, the microarray technology has been established to detect and quantify levels of acetylation. In this microarray, anti-BSA antibody was spotted onto the microarray as the capture antibody, while Bio-anti-BSA antibody and Bio-anti-Ac antibody were used as detection antibodies. By optimizing the condition, incubating protein samples, the microarray can quantify levels of acetylation and protein in parallel. This method could be applied to detect levels of acetylation on biomarkers in different stages of cancer.In the third chapter, this microarray was used to detect the levels of protein and acetylation on VEGF in serum samples. By comparing both levels of protein and acetylation on VEGF in serum from normal human and patients with hepatocellular carcinoma (HCC), this microarray can be applied to detect acetylation on biomarkers. In future work, this method will be applied to other biomarkers, such as AFP, Haptoglobin.In the forth chapter, we prepare the microfluidic bioreactor using the materials called PUF. This kind of materials enhanced the stability of protease with biological compatibility. The advantages include saving time and reagents. As there are many reactive groups, PUF is used in the biological analysis device widly.