| Proteomics is still in its infancy although great progresses have been made in proteomics during the last decade, because the extreme complexity of proteome sample and large dynamic range of protein abundance overwhelm the capability of all currently available analytical platforms. Sample pretreatment is a good approach to reduce the complexity of proteome sample and thus gain idea results from mass spectrometry-based analysis, especially for those low-abundance proteins/peptides and endogenous peptides, which may be valuable for clinical diagnosis. Therefore, new technologies for rapid enrichment of low-abundance proteins/peptides and selective separation of endogenous peptides from samples are in great demands. On the other hand, magnetic microspheres with micro-and nanometer size are gaining increasing attention due to their ease of manipulation and recovery, and functionalized magnetic microspheres have been extensively applied in cell separation, magnetically assisted drug delivery, and enzyme immobilization. In this study, we focused on preparing several kinds of novel functionalized magnetic polymer microspheres and developing a series of techniques and methods to resolve current problems in enrichment of low-abundance proteins/peptides and selective separation of endogenous peptides from samples.The whole research work in this thesis is divided into five chapters.In Chapter 1,the background and advances in proteomics research, current technologies and methods for sample pretreatment in efficient proteome and peptidome analysis, applications of functionalized magnetic polymer microspheres techniques in proteome research were summarized in details.The intention and meaning of this research work were explained.In Chapter 2, we developed oleic acid-functionalized magnetite (OA-Fe3O4) nanoparticles and C8-functionalized magnetic carbonaceous polysaccharide microspheres respectively, and applied them in enrichment of low-abundant peptides.First, the OA-Fe3O4 nanoparticles were synthesized by a one-pot method and the as-prepared nanoparticles are only about 15nm in size and possess high surface area, good dispersibility, excellent magnetic response and strong hydrophobic oleic acid group, which make them excellent adsorbents for biomacromolecules.The OA-Fe3O4 nanoparticles were successfully applied to the enrichment of low-concentration peptides from standard peptide solution, protein digest solutions and human serum. Second, biological compatible C8-functionalized magnetic carbonaceous polysaccharide (C8-F-Fe3O4@CP) microspheres with strong magnetic properties were prepared by three-step facile synthesis approach. The Fe3O4 core was firstly prepared through hydrothermal reaction, then the shell of carbonaceous polysaccharide was formed outside the Fe3O4 core. Because the shell of carbonaceous polysaccharide contain numerous hydroxyl group, carboxyl group and aldehyde group, they were then functionalized with chloro(dimethyl)octylsilane(C8), and formed C8-F-Fe3O4@CP microspheres. Thanks to the strong magnetism, many remnants hydrophilic group and surface modification with C8 groups, the process of enrichment is very simple, quick, and efficient. The resulting peptides loaded on C8-F-Fe3O4@CP microspheres can be directly analyzed by MALDI-TOF-MS without elution. The feasibility of C8-F-Fe3O4@CP microspheres was further confirmed by direct analysis of peptides in digestion products of a protein spot obtained from a 2-DE separation of human eye lens.The facile and low-cost synthesis as well as the convenient and efficient enrichment process of the novel OA-Fe3O4 nanoparticles and C8-F-Fe3O4@CP microspheres makes them promising candidates for isolation of low-concentration peptides even in complex biological samples.In Chapter 3, novel magnetic materials of C60-functionalized magnetic silica (C60-f-MS) microspheres were synthesized for the first time to enrich peptides/proteins base on the unique interaction between C60 and bio-molecules. Magnetic silica microspheres with a magnetite core and silica shell were synthesized firstly,then C60-f-MS microspheres were synthesized by radical polymerization of C60 molecules on the surface of magnetic silica microspheres。It has been demonstrated in the study, newly developed fullerene-derivatized magnetic silica materials are superior to those already available on the market. This may be arbitrated to the complex interaction between C60 and peptides, which may include hydrophobic interaction,π-bond conjugate, or some other enrichment factors. The facile and low-cost synthesis as well as the convenient and efficient enrichment process of the novel C60-f-MS microspheres makes it a promising candidate for isolation of low-concentration peptides and proteins even in complex biological samples such as urine.In Chapter 4, the functional group modified on magnetic microspheres was further improved by applying polymer of poly (methyl methacrylate)(PMMA).The sandwich structured Fe3O4@SiO2@PMMA microspheres were synthesized via combing sol-gel approach and seeded aqueous-phase radical polymerization method. PMMA polymer on the surface of the microspheres has linear hydrophobic chains, which endows Fe3O4@SiO2@PMMA microspheres good hydrophobic property, thus, Fe3O4@SiO2@PMMA microspheres could be used as hydrophobic probes toward peptide/protein. Thanks to the Fe3O4 cores in the microspheres, the enrichment process is fast without reiterative centrifugation. The co-concentration of salts can be avoided by using Fe3O4@SiO2@PMMA microspheres in the peptide absorption because of weak interaction between PMMA and hydrophilic molecular including salt. These microspheres can enrich low-concentration peptides/proteins effectively, fast and conveniently, and the resulting peptides loaded on Fe3O4@SiO2@PMMA microspheres can be directly analyzed by MALDI-TOF-MS without elution. this work can be extended to design core-shell magnetic microspheres with other polymers as shell for bioseparation applications.In Chapter 5, base on the need of endogenous peptides analysis, we develop a novel enrichment protocol for peptides by using the microspheres composed of Fe3O4@SiO2 Core and perpendicularly aligned mesoporous SiO2 shell (designated Fe3O4@nSiO2@mSiO2). The Fe3O4@nSiO2@mSiO2 microspheres possess useful magnetic responsivity which makes the process of enrichment fast and convenient. The highly ordered nanoscale pores (2 nm) and high-surface areas of the microspheres were demonstrated to have good size-exclusion effect for the adsorption of peptides. An increase of S/N (signal to noise) ratio over 100 times could be achieved by using the microspheres to enrich a standard peptide, and the application of the microspheres to enrich universal peptides was performed by using MYO tryptic digest solution. The enrichment efficiency of re-used Fe3O4@nSiO2@mSiO2 microspheres was also studied. Large-scale enrichment of endogenous peptides in rat brain extract was achieved by the microspheres. Automated nano-LC-ESI-MS/MS was applied to analyze the sample after enrichment, and 60 unique peptides were identified in total. The facile and low-cost synthesis as well as the convenient and efficient enrichment process of the novel Fe3O4@nSiO2@mSiO2 microspheres makes it a promising candidate for selectively isolation and enrichment of endogenous peptides from complex biological samples.
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