The Rapid Enzymatic Hydrolysis Of The Protein And Efficient Enrichment

Proteomics has become one of the fastest-developing areas of biological research. The commonly used strategy for protein identification consists of protein digestion and subsequent peptide-mass measurements based on mass spectrometry (MS). Protein digestion is a crucial step in the identification of proteins. Besides, peptide mapping by MALDI-TOF MS has encountered some challenges, such as difficulties in peptide preconcentration and isolation from a complex environment in proteomic analysis. Therefore, new technologies for rapid and high throughput protein digestion and selective enrichment of special subsets of peptides and proteins are in great demand. On the other hand, magnetic microsphere, an interesting advanced composite material, has received increasing attention in the past decades due to its unique physical and chemical properties and high potential applications in various fields such as cell separation, magnetically assisted drug delivery, enzyme immobilization, and protein separation.By integrating biology, chemistry and material sciences, this work aims at developing and utilizing several technologies and methods for proteolysis digestion as well as enrichment of special subsets of peptides. This dissertation is divided into four parts.In Chapter 1, advances in proteome research, global research techniques and methods of fast proteolysis and applications of functionalized magnetic polymer microspheres in biological analysis were summarized.In Chapter 2, magnetic carbonaceous (MC) microspheres prepared with a large-scale synthesis approach were developed as the novel substrate for enzyme immobilization, and the trypsin-immobilized MC microspheres were successfully applied to protein fast digestion. Firstly, MC microspheres with small size, strong magnetism, and biological compatibility, were prepared through two step solvothermal reactions. Secondly, MC microspheres surface was modified by 3-glycidoxypropyltrimethoxysilane (GLYMO). Finally, the enzyme was immobilized on the GLYMO-functionalized MC microspheres. The enzyme-immobilized magnetic microspheres were applied for fast protein digestion with microwave-assistance. Bovine serum albumin, myoglobin and cytochrome c, were used as model proteins to verify the digestion efficiency, and the digestion products were then characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with sequence coverage of 43%,90% and 77%, respectively. The enzyme-immobilized magnetic particles were also successfully applied to the analysis of human pituitary extract. After database search,485 proteins (p<0.01) were identified when the extract was digested by the microspheres. This opens a route for its future application in bottom-up proteomic analysis.In Chapter 3, laser-assisted proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly with the assistance of laser irradiation (808 nm) both in sealed transparent Eppendorf tubes and on the spots of a stainless steel MALDI plate. Besides, laser irradiation (808 nm) was employed to enhance the efficiency of in-gel tryptic digestion of proteins separated by SDS-PAGE. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of Bovine serum albumin (BSA), myoglobin and cytochrome c. It was demonstrated that laser irradiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to seconds. The in-solution digests were characterized by matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF-MS) with sequence coverage of 42%(BSA),89%(myoglobin) and 76%(cytochrome c), respectively. Besides, three replicate experiments of in-gel digestion were carried out, and the average sequence coverage of 38% for BSA and 26% for HSA from the database were obtained. The suitability of the new digestion approach to complex proteins was demonstrated by digesting rat brain. The present proteolysis protocol is simple and efficient, offering great promise for MALDI-TOF MS peptide mapping.In Chapter 4, we prepared core-shell Fe3O4@C microspheres surface-modified with fluorous functional groups and applied them to the isolation of fluorous peptides using the FSPE protocol. The efficiency and selectivity of this fluorous chemistry methodology is confirmed by MALDI-ToF MS. According to our previously method in Chapter 2, magnetite core-shell Fe3O4@C microspheres were synthesized, and further modified with 1H,1 H,2H,2H-Perfluorodecyltriethoxysilane, to present fluorous bonds on the extremities of the superparamagnetic microspheres. Because the Fe3O4@C@F microspheres possess a superparamagnetic Fe3O4 core and a fluorophilic exterior, they hold great promise for rapid magnetic separation and high absorption of fluorous species in solution-phase systems. To demonstrate the enrichment effect of Fe3O4@C@F microspheres, a model peptide (Cys-Kemptide) containing one chemically eactive cysteine residue was used. After fluorous derivatization, fluorous tags are appended and peptides are captured on the surface of Fe3O4@C@F microspheres via fluorine-fluorine interactions. The use of fluorous affinity tags makes the method suitable to various subsets of peptides from biological samples. The facile and low-cost synthesis as well as the convenient and efficient enrichment process of the novel Fe3O4@C@F microspheres makes it a promising candidate for isolation of low-concentration peptides and proteins even in complex biological samples.In summary, the main contributes of this dissertation is that we developed several techniques for rapid protein digestion and concentration of special subsets of peptides based on functional magnetic materials.