The Synthesis Of Functionalized Magnetic Materials And Their Application In Proteomics Research

With the rapid development of life science and the advent of post-genomic era, proteins as an executive of life activities function in a large number of physiology and biochemistry processes. Monitoring the dynamic change and the status of posttranslational modifications（PTMs） of proteins, and researching on the function and structure of proteins are significant for the understanding the nature of life science. Protein phosphorylation not only regulates a broad range of life activities, but also relates to the occurrence and development of many diseases. As the core technology of proteomic research, biological mass spectrometry has played an significant role and has been widely applied in phosphoproteomics study. However, it remains a major challenge for phosphoproteomic study due to the complexity of biological sample and the signal suppression of a large amount of non-specific phosphopeptides. Besides, in the mass spectrometry（MS） based bottom-up strategy, conventional in-solution digestion is obviously time-consuming and might bring in protease autolysis, which greatly affect the throughput and the coverage of protein identification. Thus, developing facile and efficient methods for protein sample pretreatment is of great significant to enhance the detection sensitivity and peptides coverage. To solve the above problems,（1） we developed two novel magnetic nanoparticles for phosphopeptide enrichment and successfully applied them to real biological samples. The results demonstrated that the newly developed methods overcame the disadvantages of low specificity of IMAC（immobilized metal ion affinity chromatography） materials and enhanced the identification efficiency of phosphopeptides.（2） We also developed a novel immobilized enzyme reactor with higher digestion efficiency based on magnetic MOFs materials and applied it to the rapid digestion of protein sample extracted from mouse oocytes.Firstly, the research status of the application of diverse materials in protein sample pretreatment was elaborated. This chapter included the application of functionalized magnetic nanoparticles in rapid enrichment of phosphopeptide and immobilized enzyme reactor, and the progress in metal organic frameworks.Secondly, based on the surface electron cloud distribution of lanthanides and their interaction with phosphate groups, we synthesized a type of mixed lanthanidesimmobilized magnetic nanoparticles Fe₃O₄@TCPP-DOTA-M³⁺ for rapid and efficient enrichment of phosphopeptide. The application of TCPP not only increased the amount of DOTA and lanthanide elements for enhanced collision rate of the materials and peptides, but also boosted the hydrophilicity of magnetic nanoparticles for higher phosphopeptide enrichment efficiency. Furthermore, macrocyclic ligand DOTA can chelate with lanthanide ions for forming extreme stable metal complex, which also stabilized magnetic nanoparticles. The results showed that 19 phosphopeptides were identified with the newly developed materials when 5 pmol tryptic digest of α-casein was used. Even with the tryptic digest of BSA over 100 times, 16 phosphopeptides were easily detected. The selectivity of phosphopeptide enrichment was obviously superior to that obtained with commercial TiO2. In a single mass spectrometric analysis, 9048 phosphopeptides corresponding to 2103 phosphoproteins were identified by the novel methods.In addition, to systematically analyse the diverse interaction of different metal ions with phosphate groups, we prepared different metal ions immobilized magnetic nanoparticles Fe₃O₄@TCPP-DOTA-Ms for selective enrichment and identification of multiple phosphorylated peptides. The results demonstrated the metal ion Tb³⁺ and Ti⁴⁺-immobilized materials had excellent enrichment efficiency and stronger adsorption for multiple phosphorylated peptides than other metal ions immobilized magnetic nanoparticles. Furthermore, the sensitivity of the transition metal ion-immobilized materials for phosphopeptide enrichment was superior to the sensitivity of the lanthanide element-chelated materials, while the specificity of the lanthanide elementchelated materials for phosphopeptide enrichment was higher than the specificity of the transition metal ion-immobilized materials. Therefore, we combined the advantage of transition metals and lanthanide elements, and successfully identified 13450 phosphopeptides corresponding to 2965 phosphoproteins. The specificity for phosphopeptide enrichment was as high as 94%. More than half of the identified unique phosphopeptides were multiple phosphorylated peptides, which was much higher than that identified by the DHB/TiO2（13.39%） method.Finally, due to reduced digestion time of immobilized enzyme reactor（IMER） and the large surface area and optional reaction sites of MOFs materials, we developed a novel immobilized enzyme reactor Fe₃O₄@DOTA-ZIF-90-trypsin through combining IMER and MOFs materials, and applied it in the proteomic analysis of protein sample extracted from mouse oocytes. The prepared magnetic MOFs with favorable magnetic response can be separated rapidly in extra magnetic field and it also possessed ultrahigh surface area, excellent structural and thermal stability. Trypsin is covalently linked to the surface of magnetic MOFs through a large amount of aldehyde groups. The results indicated that immobilized enzyme reactor had very high digestion efficiency within only 1 min with sequence coverage better than that of traditional 12 h-free trypsin digestion. Furthermore, protein sample extracted from mosue oocytes were digested by the new IMER within 20 min and in total, 8957 peptides corresponding to 1843 protein groups are identified. Compared with conventional in-solution digestion, there are nearly 40% and 67% increase in the number of identified proteins and peptides. This study not only provides a universal and effective method for magnetic MOFs materials fabrication, but also enlarges the applied scope of MOFs materials in proteomic research.