Microwave Based Magnetic Material Fixed Enzyme Hydrolysis Of New Technologies

The research work in this thesis is mainly about the new microwave-assisted protein digestion method based on functionalized magnetic nanoparticles. For the first time we developed a new facile way to synthesize magnetic nanoparticles and immobilize trypsin on them. Considering that the magnetic nanoparticles are excellent microwave absorbers, we combined the microwave-assisted protein digestion method with the immobilized trypsin, which greatly increase the digestion efficiency. Compared with the traditional in-solution digestion method, this new digestion method shortened the process from 12h to 15s, and the digestion efficiency is better than or equivalent with the traditional in-solution method. With the help of the microwave irradiation, even protein ofμg quantity can be digested efficiently. To further verify its feasibility for real proteome, the whole protein extract of rat liver was digested by this method. The produced digests in 15s were analyzed by LC-MS/MS directly and lots of proteins were successfully identified. Besides, because the trypsin-immobilized magnetic nanoparticles can be easily retained by an external magnet, the immobilized trypsin can be recycled and reused. The whole research work in this thesis is divided into four chapters.In Chapter 1, the background and purpose of this research are demonstrated. The fast development of proteome technology and importance of protein digestion in this area are introduced. Then I introduced the new achievements in the development of digestion approaches, mainly including immobilized trypsin and microwave-assisted protein digestion. Using immobilized enzyme to replace free enzyme is a trend of new technologies in digestion method development. Microwave-assisted protein digestion is also attracting more and more attention due to its easy manipulation and high efficiency. The research work about the combination of the two is reported little, and mainly focusing on standard proteins.In Chapter 2, new microwave-assisted protein digestion method was developed by easily prepared trypsin-immobilized magnetic microspheres. Magnetic microspheres with small size were synthesized and modified by 3-glycidoxypropyltrimethoxysilane (GLYMO). Trypsin was immobilized onto magnetic microspheres through only a one-step reaction of its amine group with GLYMO. When these easily prepared trypsin-immobilized magnetic microspheres were applied in microwave-assisted protein digestion, the magnetic microspheres not only functionalized as substrate for trypsin immobilization, but also as an excellent microwave absorber and thus improved the efficiency of microwave-assisted digestion greatly. Digests produced in microwave-assisted protein digestion were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Cytochrome c was used as a model protein to verify its digestion efficiency. Peptide fragments produced in 15 s could be confidently identified by MALDI-TOF MS and better digestion efficiency was obtained comparing to conventional in-solution digestion (12 h). Besides, with an external magnet, trypsin could be used repeatedly and at the same time no contaminants were introduced into the sample solution. It was verified that the enzyme maintained high activity after seven runs. Furthermore, reversed-phase liquid chromatography (RPLC) fractions of rat liver extract were also successfully processed using this novel method.In Chapter 3, Fe₃O₄-GLYMO microspheres developed in Chapter 2 were improved for trypsin-immobilization. Considering the lower enzyme upload, we increased the functionalized groups on the surface of magnetic microspheres by covering silica around Fe₃O₄. More Si-OH groups were formed after this step and thus increase the enzyme upload in the following step. UV-absorb was used to calculate the upload and the results showed that it had increased as much as 3to 4 times as before. The covalent interaction between enzyme and magnetic microspheres was verified by SDS-PAGE. Besides, after silica covering, the formed silica magnetic microspheres owned more solubility. Three standard proteins were used to optimize the digestion conditions and the MALDI mass spectra displayed successful identification of the proteins digested in 15s microwave incubation. To further verify its worth for real proteome application, whole rat liver extract was digested by this 15s method and analyzed by liquid chromatographyelectrospray-tandem mass spectrometry (LC-ESI-MS/MS), comparable digestion efficiency was observed with typical in-solution digestion but the incubation time was largely shortened. This new microwave-assisted digestion method will hasten the application of the proteome technique to biomedical and clinical research.In Chapter 4, magnetic carrier was further improved by applying amine-group magnetic nanoparticles in microwave-assisted protein digestion. Comparing to magnetic silica microspheres, amine-group magnetic nanoparticles have higher surface/volume ratio and the producing process was much more time and labor saving. In the following similar microwave-assisted process and proteome analysis process, trypsin immobilized on amine-group magnetic nanoparticles displayed high efficiency for both standard proteins and real proteome. More detailed comparing work to traditional in-solution digestion was done and the result was much encouraging.