Development Of Novel Isolation And Identification Approaches In Proteomics Analysis Using Functional Magnetic Technologies

Based on the proteome research background and the development trend of polymer magnetic microspheres, the research interest of this work focused on preparing several kinds of novel functionalized magnetic polymer microspheres and developing a series of techniques and methods to resolve current problems in the separation and concentration of low-abundance and post-translational proteins. This dissertation is divided into five parts.In chapter 1, advances in current protein separation and concentration techniques, applications of functionalized magnetic polymer microspheres techniques and application of functional materials in proteome research were summarized in details. The intention and meaning of this dissertation were explained.In chapter 2, magnetite particles were synthesized via solvothermal reaction; and then magnetite microspheres were coated with silica through sol-gel methd; subsequent modified with chloro(dimethyl) octylsilane;thus a novel C8-functionalized magnetic polymer microsphere was prepared. With the characterization of vibrating-sample magnetometer, FT-IR, TEM, SEM and Thermogravimetric analysis, the resulting C8 functionalized magnetic silica microspheres exhibited well-defined magnetite-core-silica-shell structure and possess high content of magnetite, which endow them with high dispersiblity and excellent magnetic responsibility. As a result of their excellent magnetic property, the synthesized C8 -functionalized magnetic silica microspheres were successfully applied for convenient, fast and efficient enrichment of low-abundance peptides from tryptic protein digest and human serum via a direct MALDI-TOF mass spectrometry analysis. The signal intensity could be improved by at least two orders of magnitude.Even in high salt concentration solution, the peptides could also be isolated effectively. The experiment results were almost the same with the results gained using the C8 -functionalized magnetic silica from Bruker Company in the reference. The facile synthesis and the convenient and efficient enrichment process of the novel C8-functionalize microspheres make it promising candidate for isolation of peptides even in complex biological samples.Chapter 3 presents a facile way to modify the surface of magnetic silica microspheres and then immobilize Fe³⁺ ions on their surface based on the principle of IMAC, thus Fe³⁺-immobilized magnetic silica micorspheres (Fe³⁺-MS) were prepared. We confirmed them with FT-IR, TEM and SEM. Because of high magnetic responsivity to magnetic field and the introduction of Fe³⁺ on the smooth silica coatings, the prepared Fe³⁺-MS could be applied to efficiently and conveniently enrich trace peptides with a help of applied magnetic field. The Fe³⁺-MS showed good selectivity to phosphopeptides and the S/N signal could be improved by at least one order of magnitude. The experimental conditions for the enrichement of phosphopeptides were optimized. In more complex sample, phosphopeptides could also be selectively enriched. The captured phosphopeptides could also be direct analysis by MALDI-TOF MS avoiding the sample loss during elution from the material.The Fe³⁺-MS were applied to capture the phosphopeptides in complex biological samples. Some phosphopeptides in normal human serum were enriched and their composition and phosphotylated site were determined by MALDI-TOF MS/MS. It was also applied to isolate the phosphopeptieds in trypsin digest of rat liver protein lysate.we took two technical routes: (1) isolated the phosphopeptieds in trypsin digest of rat liver protein lysate, then analyzed the eluted sample by nanoLC- LTQ MS/MS and 66 phosphopeptides were successfully identified. (2) separated the trypsin digest of rat liver protein lysate by RPLC and 29 fractions are collected. Thus the complexity of the fraction was lowered. Then isolated the phosphopeptieds from each fraction and then analyzed the elution by MALDI TOF MS.In addition to above work, we developed a more convenient method to synthesize a novel functional magnetic material for phosphopeptides isolation. Amine-magnetite nanoparticles were synthesized by solvothermal reaction and were further converted into carboxy-magnetite nanoparticles through two-step amidation reaction. Then, Fe(III),Al(III),Ga(III),In(III),Zr(IV),Ce(III) ions were immobilized on the surface of the nanoparticles via chelation respectively which were confirmed by characterization with FT-IR, TEM, SEM and EDXA analysis. All of The six materials showed good selectivities to phosphopeptides, especially Fe(III),Al(III),Ga(III) and Zr(IV).In chapter 4, we present an innovative approach for the synthesis of TiO₂-coated magnetite microspheres (Fe₃O₄@TiO₂ microspheres) with well-defined core-shell structure, which were confirmed by characterization with FT-IR, TEM, SEM and EDXA analysis. This material enriched phosphopeptides by the affinity between TiO₂ and phosphopeptides and was more stable than the IMAC materials. By using the Fe₃O₄@TiO₂ microspheres with the assistance of applied magnetic field, we demonstrate the high efficiency of the smart application of these microspheres as specific capture of phosphopeptides for MALDI-TOF MS analysis. The experimental conditions for the enrichement of phosphopeptides were optimized. In more complex sample, phosphopeptides could also be selectively enriched effectively.The process of enrichment is very facile, efficient and highly selective. The peptides adsorbed on the Fe₃O₄@TiO₂ core-shell microspheres can be directly analyzed by MALDI-TOF-MS analysis without elution from the Fe₃O₄@TiO₂ core/shell microspheres.The Fe₃O₄@TiO₂ core-shell microspheres were applied to capture the phosphopeptides in normal human serum. Two phosphopeptides were enriched and their composition and phosphotylated site were determined by MALDI-TOF MS/MS. It was also applied to isolate the phosphopeptieds in trypsin digest of rat liver protein lysate and then the eluted sample was analyze by nanoLC- LTQ MS/MS and 79 phosphopeptides were successfully identified. These results are expected to open up a new possibility for the enrichment of phosphopeptides. Moreover, the versatile, low-cost and reproducible synthesis method could extend to other core-shell structured microspheres of metal oxides.In chapter 5, the boronic acid or Con A modified magnetic nanopartiles were synthesize and applied to selectively enrich glycoprotein from protein mixture.the experimnt result indicated that the boronic acid modified magnetic nanopartiles effectively captured the glycoprotein. In addition, the digest conditions of glycoprotein were modified and glycopeptides were separated from the digest mixture by RPLC and then confirmed by MALDI-TOF-MS.In summary, the main contributes of this dissertation is to develop several functional magnetic materials and found several new separation methods for low-abundance and post-translational proteins. We aim at exploring and finding out new techniques in the separtion and concentration of proteome research fields, so that more breakthroughs can be obtained in the proteome research study.