| Now the peptidome research has attracted increasing attention in academia in virtue of simplicity of sample preparation and possibility of recording current physiological states of the body as metabolic products of proteins.Circulating protein fragments generated in the body fluid or tissues may probably reflect biological events and provide emerging information for clinical diagnosis. And the real role of the peptidome needs to be further studied. Peptidome refers all of low-molecular-weight (LMW) proteins, for exsample endogenous peptides, in complex biological samples such as body fluids, cell lysate and tissue extract. Therefore, peptidomics is defined as systematic, holistic, qualitative and quantitative analysis of the endogenous peptides and small proteins in biological sample at a defined time and location. In apeptidome study, endogenous peptides are recently shown to contain protential biomarkers for recording the physiological and pathological state of human beings, which are more sensitive and specific for clinical applications. For peptidome analysis, the important step is to extract the peptidome fromcomplex biological samples. And high-abundance proteins in complex biological samples often cover the signal ofpeptides in MS analysis, so the key issue is to isolate the small peptides from the high concentration of large protein samples. For this purpose, magnetic particles-based solid phase extraction become a facile and efficient method for selective enrichment of endogenous peptides. In this study, we focus on synthesizing novel functional magnetic mesoporous microspheres and developing efficient techniques for selective enrichment of LMW endogenous peptides in peptidome analysis. The whole research work is divided into four chapters.In chaper 1, development of proteomics and peptidomics, current methods of sample preparation in proteome and peptidome analyisis, magnetic particles-based solid phase extraction techniques in peptidomics, and applications of functionalized magnetic nanoparticles in proteome research were summarized. Finally, the purport and target were discussed.In chapter 2, Cu2+-immobilized core-shell magnetic mesoporous silica microspheres were facilely synthesized through a surfactant-templated sol-gel process and subsequent chelation of Cu2+ ions. The obtained microspheres possess high magnetization saturation (43.6 emu/g), large surface areas (85 m2/g), accessible uniform mesopores of 3.3 nm as well as high-density immobilized Cu2+ ions. The microspheres were successfully applied to enrich hydrophobic and hydrophilic peptides from standard peptides solution and tryptic protein digest solution. More importantly, it is demonstrated that the functional microspheres can be used to selectively capture peptides from complex sample systems (such as human serum and urine) with excluding the large proteins. Because the microspheres combine the merits of magnetic property of Fe3O4, porosity of mesoporous silica and specific affinity of Cu2+ ions toward peptides, the process of selective peptides enrichment is very convenient and efficient.In chapter 3, novel magnetic mesoporous silica microspheres with C8-modified interior pore-walls were synthesized through a facile one-pot sol-gel coating strategy and were successfully applied to selective enrichment of endogenous peptides in biological samples for peptidome analysis. Since the C8 moiety was introduced during surfactant templated sol-gel process, the hydrophobic C8 chains anchored on the pore-walls would not change the porosity of mesoporous silica microspheres after removal of surfactant template, resulting in the ordered narrow pore with size of. 3.4 nm. Thanks to the abundant surface silanol groups, functional C8 groups and the strong magnetic responsivity, the core-shell C8-Fe3O4@mSiO2 microspheres display good dispersibility, fast magnetic response, which makes the process of peptide enrichment very simple, quick and efficient. More importantly, the C8-Fe3O4@mSiO2 microspheres were demonstrated to selectively capture endogenous peptides from complex samples, such as human serum, containing large proteins based on size exclusion effect. Finally, the C8-Fe3O4@mSiO2 microspheres were successfully applied to selectively enrich endogenous peptides from mouse brain extract followed by automated nano-LC-ESI-MS/MS analysis. The number of peptides identified is up to 267, which is 3.45 times more than that of previous work, as a result of the distinguished size-exclusion effect, hydrophobic interaction and good dispersivity. In comparison with previous work, more peptides, molecular weight of which are lower than 1200 Da could be enriched and identified after treatment by C8-Fe3O4@mSiO2 microspheres. In addition, based on the size-exclusion effect of mesopores, about 85% of identified peptides are those that have shorter than 20 amino acid at length. Considering the result above, the facile and low-cost synthesis as well as the convenient and efficient extraction of peptides before MS analysis indicates the C8-Fe3O4@mSiO2 microspheres would be a potential candidate for endogenous peptides enrichment and biomarkers discovery in peptidome analysis. In chapter 4, calcined MWCNTs/Fe3O4-@mSiO2 were facilely synthesized through a self-assemble process to hybrid MSCNTs with Fe3O4 and afterwards a surfactant-templated sol-gel process to coat mesoporous silica shell. The obtained materials have (161 m2/g), easily accessible uniform mesopores with 3.69 nm. And the materials were also applied successfully to the enrichment for peptides in standard peptide solution and tryptic protein digest solution, Which proved a convenient and efficient process. Peptides can be easily captured in solution as a result of ordered mesoporous silica. Meanwhile, c-MWCNTs/Fe3O4-@mSiO2 materials reveal the size-exclusion in enrichment of peptides from complex biosamples, such as mouse brain, because of the high magnetic responsivity and uniform mesopores. After database searching,98 peptides were detected from the eluent with treatment by c-MWCNTs/Fe3O4-@mSiO2, and MWs of identified peptides centralize on the range from 1200 Da to 2600 Da. Another unique character of c-MWCNTs/Fe3O4-@mSiO2 materials is infrared irradiation adsorption which can help to elute enriched peptides from materials in a short time. This technique is expected to applied in the enrichment of endogenous peptides in biosamples, as well as a new application of carbon nanotubes and magnetic mesoporous materials.In summary, a series of techniques and methods based on magnetic particles for endogenous peptides enrichment were developed. Novel functionalized magnetic mesoporous microsphere were successfully synthesized and applied to selective enrichment of endogenous peptides in complex biological samples. The facile and low-cost synthesis as well as the convenient and efficient extraction of peptides before MS analysis indicates the functionalized magnetic mesoporous microspheres would be potential candidates for endogenous peptides enrichment and bio markers discovery in peptidome analysis. |
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