Synthesis Of Functionalized Nanocomposites By "Thoil-ene" Click Chemistry And Their Applications In Enrichment Of Post-translational Proteins

As a branch of proteomices,post-translational proteomices have attracted great attention.At present,the main bottleneck of post-translational proteome research is to explore new approaches for enrichment of low-abundance post-translational proteinsfrom complex biological samples."Thoil-ene" reaction has been widely used in medicinal chemistry,biological sciences and chromatographic stationary phases due to the merits of simplicity,high.efficiency,high selectivity and high conversion under mild conditions.In current research,combining the unique properties of nanomaterials with the "thoil-ene" click reaction and affinity chromatography,we developed several methods for synthesis of functionalized silica nanocomposites,which were further applied to the separation and enrichment of glycoproteins and phosphoproteins from the complex biological samples.The contents of this paper include:In chapter 1,ageneral review of the separation and enrichment methods of the post-translational proteomices,as well as its advances was introduced,specially focusing on description of the separation and enrichment methods of glycoproteins and phosphoproteins based on nanomaterials.Inaddition,the significance and content of this thesis were also briefly presented.In chapter 2,A facile method was developed for synthesis of Ti4+ immobilized silica nanocomposites(SiO2-Ti4+)by "thiol-ene" click reactionin one-potprocess,where SiO2 was prepared by the sol-gel process and vinylphosphonicacid(VPA)as a functional monomers was coated at the surface of the silica nanoparticles by the"thiol-ene",reation,and then Ti4+ was immobilizied by self-assembly.The morphology and structure property of the resultant SiO2-Ti4+ were characterized by Transmission electronic microscopy(TEM),Fourier transform infrared spectroscopy(FT-IR)and X-ray photoelectron spectroscopy(XPS)and so on.The adsorption behavior of the SiO2-Ti4+ toward phosphoproteins was investigated.Under the optimized conditions,the SiO2-Ti4+ showed higher recognition ability towards phosphoproteins(a-casein(12.6 ?mol/g)than non-phosphoproteins(horseradishperoxidase(1.12 ?mol/g)).Inaddition,the practicality of the SiO2-Ti4+was further assayed by selective enrichment of phosphoproteins from milk samples.In chapter 3,based on the principle of boronate affinity inter action,a boronate affinity silica nanocomposites were synthesized by"thiol-ene"click reaction and applied to glycoproteins recognition.The morphology and structure properties of the resultant boronate affinity silica nanocomposites were characterized by Transmission electronic microscopy(TEM),Fourier transform infrared spectroscopy(FT-IR)and Thermo gravimetric analyzer(TG)andsoon.The adsorption behavior of the silica nanocomposites toward glycoproteins was investigated.Under the optimized conditions,the silica nanocomposites showed excellent recognition ability towards glycoproteins(ovalbumin(6.32?mol/g),compared to non-glycoproteins(bovine serumalbumin(0.83 ?mol/g)).Inaddition,the feasibility of boronate affinity silica nanocomposites was further assayed by selective enrichment of glycoproteins from the egg white samples.In chapter 4,a polydopamine-coated molecularly imprinted silica nanoparticles(MIP)was prepared by "thiol-ene" click reaction using SiO2 as core,3-acrylamidophenyl boronicacid(AAPBA)as functional monomer,horseradishperoxidase(HRP)as glycoprotein template.Well defined core-shell structure of MIP was obtained after self-polymerization ofdopamine(DA)on the surface of HRP-immobilized silicaNPs.The polymerization conditions and adsorption behavior were investigated in detail in order to obtain the highest selectivity and binding capacity.In the optimized conditions,the MIP showed higher binding affinity toward the template than non-imprinted(NIP)nanoparticles,the crossponding adsorption capacity(Q)and imprinted factor(a)reached 0.58 ?mol/g and 2.76,respectively.The specificity for HRP recognition wase valuated with competitive experiments,and the result indicated the imprinted nanoparticles had a higher selectivity for thet emplate.Besides,The resultant MIP could not only differentiate the template HRP from protein mixture,but also selective enrichment of trace HRP from human serum.Inaddition,The stability and regeneration were also investigated,which indicated the imprinted silica NPs had excellentre usability.