Synthesis Of Functional Magnetic Nanomaterials For Protein/Peptide Separation And Enrichment

Based on the proteome research background and the development trend of magnetic nanomaterials,the research of this work focused on preparing several kinds of novel biofunctional magnetic nanomaterials and developing a series of techniques and methods to resolve current problems in the separation and enrichment of glycoproteins/glycopeptides and histidine-rich proteins.There are six chapters in the thesis.In chapter 1,we give a detailed review on the advances in current protein separation and analysis techniques,the synthesis of functionalized magnetic nanomaterials,and the application of biofunctional magnetic nanomaterials in proteome research.In addition,the intention and meaning of this dissertation were explained.In chapter 2,we developed a facile one-pot method for the synthesis of phenylboronic acid（PBA）functionalized core-shell magnetic nanoparticles（MNPs）.The morphology,adsorption,and recognition properties of the resultant MNPs were investigated.The resulting MNPs demonstrated high binding capacity and selectivity for glycoproteins（320 mg/g for ovalbumin and 165 mg/g for mucin）and ability in selective enrichment of glycoproteins from human serum.In chapter 3,we demonstrated a method to synthesize PBA functionalized Fe₃O₄@polydopamine magnetic microspheres via the combination of mussel-inspired polydopamine coating and click chemistry.Uniform-size and core-shell structured magnetic microspheres with a core diameter of～240 nm and a shell thickness of～13 nm were obtained.Adsorption experiments,adsorption experiments and mass spectrometry analysis demonstrated that the obtained magnetic microspheres had much high binding capacity and selectivity for glycoproteins/glycopeptides compared to nonglycoproteins/nonglycopeptides.In addition,the practicability of the magnetic microspheres was further assessed by selective capture of glycoproteins from human serum.In chapter 4,we reported the click synthesis of glucose-functionalized magnetic mesoporous nanoparticles（MVMNs）,which hold the attractive features of well-defined core/shell structure,high specific surface area（324 m2/g）,narrow pore size distribution（2.2 nm）and high magnetic responsivity（69.1 emu g-1）.The hydrophilic MMNs possessed high selectivity for glycopeptides at the digested mixtures of horseradish peroxidase,myoglobin andβ-casein at molar ratio of 1:1:10,large enrichment capacity（as high as 250 mg/g）,high sensitivity（50 fmol）,excellent speed（5 min for enrichment）and high recovery of glycopeptides（as high as 94.6%）.Additionally,the MMNs exhibited excellent performance in enrichment of N-linked glycans from the digested human serum.In chapter 5,we present a facile ligand-free method for the rapid and highly specific separation of histidine（His）-rich proteins using CuFe₂O₄ magnetic nanocrystal clusters（MNCs）.Monodispersed CuFe₂O₄ MNCs were synthesized via a simple and economical one-pot hydrothermal process.The resulting MNCs were characterized in detail.The measurements indicated that the MNCs exhibited good dispersion,high crystallinity,high saturation magnetization（45.1 emu g-1）,and superparamagnetic properties.The selectivity and binding capacity of CuFe₂O₄ MNCs were evaluated using a His-rich protein（bovine haemoglobin）and other proteins containing fewer surface-exposed His residues as model samples.The most distinct feature of the CuFe₂O₄ MNCs is the high haemoglobin binding capacity（4475 mg g-1）.In addition,the CuFe₂O₄ MNCs can be successfully employed to selectively bind and remove abundant haemoglobin from human blood samples.The good results demonstrate the potential of CuFe₂O₄ MNCs in the separation of His-rich proteins.In chapter 6,we described a novel magnetic zeolitic imidazolate framework-8（Fe₃O₄@ZIF-8）based metal affinity platform for the rapid and highly specific separation of histidine-rich proteins.The coating of ZIF-8 layer on Fe₃O₄ core was performed in aqueous solution at room temperature and merely took 10 minutes.The monodisperse Fe₃O₄@ZIF-8 display superparamagnetism with a saturation magnetization value of 47.9 emu g-1,and possess large external surface area of 131.0 m2 g-1.Fe₃O₄@ZIF-8 exhibited large adsorption capacity for BHb（>6000 mg g-1）.Moreover,Fe₃O₄@ZIF-8 showed excellent recyclability（more than 10 times）with high recovery（88.4%）.In addition,Fe₃O₄@ZIF-8 can be used to selectively separate hemoglobin from protein mixture and human blood samples.The good results demonstrate the potential of Fe₃O₄@ZIF-8 in the separation of histidine-rich proteins.