Study On The Method Of Enzyme Enrichment And Protein Efficient Enzymatic Hydrolysis Based On Functional Nanomaterials

Protein is a direct manifestation of biological life phenomenon, its research has very important meanings.If we can study clearly the structure and function of protein, understand the interaction between protein and protein and know the differences of protein expression in different environment and the function in the specific channel, thus we can explain some changes in physiological or pathological conditions. Therefore, it is necessary to develop new techniques and methodologies for better solution of proteome research and provide important basis for pathology, physiology and pharmacology.In the study of proteome, developing an effective and rapid digest method is one of the most important subjects. And the difficulties of peptidomics research is to find an appropriate method for effective enrichment, thus we can avoid some disadvantages, such as high concentrations of salt,high-abundant protein and so on.In recent years, with the rapid development of functional nanomaterials, it has been used in various fields. Compared with other materials, they have a larger specific surface area and specific functions, make it suitable for biomedical research area. Moreover, functionalized materials with specific modification can be effectively applied in proteome and peptidomics research and simplified the experimental procedure.Based on proteome research and the developmnet of functional nanomaterials, in our work, we focused on the efficient digestion and selective enrichment, thus we developed a series of novel techniques and methods to resolve these problems. The feasibility of these techniques and methods was proved with complex biological samples and they showed great potential in proteome research. Specific content as follows:In chapter 1, we mainly summarized the importance of proteome research and introduced some mainly techniques and methods foe protein analysis. Next, we reviewed the application of functional nanomaterials in proteome research, including immobilized enzyme technologies and different digestion methods. Moreover, we introduced some novel techniques about the application of functional nanomaterials in peptidomics research. Finally, we put forward the purpose and significance of the thesis topic.In chapter 2, graphene for the first time was applied to detect glutathione by MALDI-TOF-MS and stable analysis was achieved with less background inference even at the concentration of 0.625 ng/μL. Compared with other methods, our work can simplify sample preprocessing steps, the efficacy of separation was much higher than traditional methods. The proposed method has been successfully applied to detect the GSH in mouse liver extraction. Thus, this kind of rapid, simple, and direct approach shows the great potentials of detection of biologically important thiols in biochemical and biomedical research.In chapter 3, a new Tips with gold modified polymer has been developed, the simple, self-made and extremely economical Tips were successfully applied to capture the cysteine-containing peptides due to the strong interaction. We can make one Tip in 30 seconds, and depending on the application, different thickness and diameter can be chosen. Experimental results demonstrated that it has high specificity and selectivity for cysteine-containing peptides in the presence of a 100-fold excess peptides containing no thiol over cysteine-containing peptides. All the work can be finished in 3 minutes. The studies carried out to date show that the new Tips have minimal cost, perfect reusability and excellent selectivity. Moreover, the Tips are well-suited as a universal sample pretreatment system for proteomics.In chapter 4, Fe3O4@Graphene Oxide@Trypsin was synthesized, Firstly, Fe3O4@Graphene Oxide was prepared via a solvothermal reaction, and then it was applied to immobilize trypsin. Fourier transform infrared spectroscopy (FTIR) was used to characterize both the Fe3O4@Graphene Oxide and Fe3O4@Graphene Oxide@Trypsin composites, the results showed the successful modification on the surface of Fe3O4@Graphene Oxide. Due to the large specific surface area and strong hydrophilicity of Graphene Oxide, the binding capacity achieved to be 395 μg/mg, which is much higher than many other nanoparticales. Moreover, it had great digestion performance with laser-accelerated digestion for only 5 s. There is no obvious difference with traditional aqueous digestion. In addition, we used this method to digest complex protein sample extracted from rat brain, the results showed that within only 15 min, we can detect the 1080 protein groups in the rat brain extract, thus further proving the digestion efficiency of our composites. Therefore, we have provided a new efficient and rapid digestion technology.