Study On Separation And Analysis Of Acetylated Protein

The word "proteome" was firstly mentioned by Marc Wilkins from Macquaie University in Australia in 1994. It means a full set of protein contained in a genome, a kind of species, cell or tissue. Proteome science is a new-rising subject in which high-throughput and high-sensitivity techniques are utilized to systematically study the proteins in biological systems and their activity rules. The research includes protein identification, post-transcriptional modification, and protein function confirmation and so on. Particularly, the research on tumor cell growth, differentiation and tumor formation is getting deeper and deeper in recent years, which offers important evidences for early diagnosis of tumor, discovery of drug targets, judgment of treatment results and prognosis effect.Histones are basic proteins which exist with DNA in eukaryotic cell nucleus. The imbalance of their acetylation is closely related to the occurrence of cancer. It has been reported that acetylation level of histone H3 has important clinical diagnosis function of prostate cancer, breast cancer, lung cancer, kidney cancer, such as in prediction of tumor recurrence, tumor-related phenotypes, prognostic factors, and prognosis of patients. Primary liver cancer is a common malignant tumor, with a fatality rate ranking third among all the types of cancers in the world. Hepatocellular carcinoma (HCC) accounts for 90 percent of primary liver cancer in China. Up to now, there is no research or report about acetylation locus and level of histone H3 in liver cancer. The status of quantitative proteomics has been increasingly important in the physiological and pathological study. In the field of quantitative proteomics based on mass spectrometry, isotope labeling, compared to other technologies, has the characteristics of high efficiency, high precision and high sequence coverage, but expensive isotope reagent and complex marker technology limit its application. In this part, we apply a relatively cheaper esterification reaction to this field. Methylation markers occur in amino acid group (Asp and Glu) and C on the side, making the marker loci produce quality difference of 3.0188 Da. Good linearity and repeatability means that methylation reaction can be well combined with LTQ-Orbitrap technology. Through chromatographic separation, combination of methylation quantification and LTQ-Orbitrap technology, the experience carries out quantitative analyses on histone H3 acetylation modification site of normal liver L02 cells and non-transfer HepG2 liver cancer cells, liver cancer transfer cell 97H and non-transfer liver cancer cell HepG2, high transfer potential cell LM3 and low transfer potential cell 97L. Acetylation peptides of which quantitative ratio is greater than 2 or less than 0.5 are chosen as the peptides that have changed, then quantitative information of 10 acetylation and methylation modification peptides is obtained. Compared to histone H3 post-modification site in L02,50% peptides content in HepG2 has dropped significantly. Compared to histone H3 post-modification site in HepG2, the content of eight peptides in ten peptides identified in 97H has obviously decreased. Compared to low transfer potential liver cancer cell 97L, the content of six peptides in high transfer potential liver cancer cell LM3 has obviously decreased. In addition, in those five groups of cell lines, acetylation peptides K* SAPSTGGVK ("* "on behalf of acetylated modification) of isomer H3.3 of histone H3 are identified for the first time. This method can be applied to the histone post-transcriptional modification study and quantitative analysis, the results provide more information for deep research of liver cancer and other cancers histon post-transcriptional modification.Efficient enrichment method is an indispensable step in large-scale identification of acetylated modification. Traditional acetylated protein identification methods include radioactive and immune affinity tests, but these methods can only detect the existence of acetylated protein, specific acetylated modification site information cannot be achieved. The combination of acetylation enrichment technology and mass spectrometry identification technology is also the mainstream method in the study of acetylation, which can not only detect post-transcriptional modification protein, but also can get specific modification site information, while using lysine acetylation antibody to gather acetylated modification is also the main enrichment method so far. Microfluidic chip has been widely used in the study of proteomics analysis because of its fast analysis speed, high throughput, small sample loss, and low cost, and it has increasingly shows its superior performance. In a multitude of microfluidic chip materials, PDMS has been widely applied, especially connected to a pump system to achieve the integration of chemical reaction and biological processes and microchip analysis. Due to the hydrophobicity of polymer, PDMS is easy to adsorb proteins, bacteria and cells non-specifically, which affects the biological analysis efficiency of chip. Meanwhile, the polymer surface energy is low and lack sufficient reactive group needed by functionalization reaction. Therefore, material surface should be modified when PDMS is used to produce functional cell chips. In recent years, with the development of separation and enrichment technology, a large number of acetylated protein and sites have been identified to provide more information for the research of its function. Because of the low concentration of acetylated protein in the cell, the signal will be affected by a lot of non-acetylation protein in the appraisal, which brings a big challenge to the acetylated protein identification and function study. In this research work, the microfluidic chip is combined with Protein A/G to directionally immobilize antibody for the first time. Acetylated antibody is fixed in homemade microfluidic chip through the Protein A/G to gather acetylated protein and acetylated peptides, and MALDI-MS is used to identify the acetylated protein and acetylated loci. The results showed that Protein A/G could well couple with the constant area of antibody. This strategy can keep the activity of antibody very well, and can greatly improve the efficiency and specificity of antibody with antigen. The enrichment efficiency of acetylated Protein in this method can reach 82.4%, compared with before enrichment. Hence, we can get more information on the acetylation of peptides in mass spectrum graph after enrichment. This method has mild reaction condition, simple operation steps, and good repeatability, opening up a new route for the enrichment and identification of protein acetylation.In this work, we modified the hydrophobicity surface of chip to hydrophilic surface to obtain good biological activity, and then Protein A/G Beads were immobilized on the chip surface through the positive and negative electrical properties. The main advantages of this chip lie in:1) fixed direction of antibody is not random, Protein A/G can be combined with the constant area specificity of antibody, which can not only keep the activity of antibody very well, but also can improve the combination efficiency and specificity of antigen; 2) using microfluidic chip to fix antibody through Protein A/G, which can effectively reduce the amount of antibody and antigen, and can achieve good detection effect; 3) mild reaction conditions and simple steps save the experiment time and cost.In the study, we compare chip immobilized antibody method with common immune precipitation (IP) method. Using chip-fixed antibody to gather acetylated protein needs less samples and the enrichment efficiency is much higher than the traditional immune precipitation method. IP is mainly used for qualitative and semi-quantitative detection of antigen or antibody, which uses combination of antigen protein and the specificity of the antibodies, as well as the phenomenon of bacterial protein specificity combined with Fc immunoglobulin fragments. Thus an immunological method of separation of antigen is developed. Commonly used immune precipitation method is the agarose beads method. This method can efficiently gather antigen, obtain antigen-antibody complex by centrifugation, and through further steps, such as elution and identification, target protein is obtained and its properties are analyzed. However, only is high-speed centrifugal to separate bead from the system, which is time-consuming and complicated, and difficult to ensure no damage of settle beads in the process of centrifugal drain and leaving supernatant. Using fixed antibody concentration acetylated protein chip need less amount of samples, and enrichment efficiency is much higher than the traditional immune precipitation Our new method can well avoid these drawbacks of IP to save detection time, simplify detection steps and improve detection efficiency. This method is worth popularizing in protein research.