Study On The Selective Enrichment Of Phosphorylated Proteins/Phosphopeptides By Immobilized Ti⁴⁺ Magnetic Nanoparticles

Phosphorylation is one of the important behaviors of protein post-translational modification and plays an indispensable role in life regulation.Abnormal phosphorylated proteins/phosphopeptides are often biomarkers for many diseases,so the study of phosphorylated proteomics is particularly important.Mass spectrometry is often used for qualitative analysis of phosphorylated proteins/phosphopeptides.Due to the low throughput of phosphorylated proteins/phosphopeptides,it is difficult to ionize and easily be interfered with signals from non-phosphorylated proteins/non-phosphopeptides,and directly analysis is more difficult.To separate and enrich the phosphorylated protein/phosphopeptide before mass spectrometry analysis is necessary.Therefore,developing a simple and efficient synthetic adsorption material for the phosphorylated protein/phosphopeptide enrichment is very urgent.In order to achieve the separation and enrichment of the target analytes,based on the previous work of our research group,this thesis has made innovations based on the previous work of the research group,that is,a simpler chelating ligand alendronate sodium is selected,and ferric oxide?Fe₃O₄?magnetic nanoparticles as the matrix are designed to two different types of adsorbents whose performance are further research.1. The solvothermal method was used to prepare Fe₃O₄ magnetic nanoparticles,and their surface was coated with dopamine,then the Fe₃O₄@PDA magnetic nanoparticles were used as the secondary reaction platform to modify alendronate sodium,and finally the target adsorbent was chelated with Ti⁴⁺.The adsorbents were characterized by fourier-transform infrared,X-ray photoelectron spectroscopy,X-ray powder diffraction and transmission electron microscopy.The adsorption performance and kinetic behavior of the adsorbent for standard proteins??-casein and ovalbumin?were studied.The influence of the solution p H and salt concentration on the adsorbent was investigated,which showed that the adsorbent was adsorbed by metal coordination,and the adsorption behavior was more consistent with langmuir model and quasi-second-order kinetic model.At the same time,it was compared to the control adsorbent Fe₃O₄@PDA-Ti⁴⁺,the research focused on the binding strength of the adsorbent to the target proteins,the adsorption capacity and the stability of the adsorbent during the adsorption process(the amount of Ti⁴⁺loss).Finally,the adsorbent was successfully used to selectively separate and enrich OVA and?-casein from actual samples of eggs and milk,respectively,indicating that the adsorbents prepared have potential application value in biological application in this thesis.2. The solvothermal method was used to prepare Fe₃O₄ magnetic nanoparticles,which was coated with a layer of silica to improve the hydrophilicity of the material. Finally,the target adsorbent was prepared by chelating Ti⁴⁺.Scanning electron microscope,infrared spectroscopy and thermogravimetric analysis were used to characterize the adsorbent.The performance of the adsorbent on the phosphopeptide in?-casein hydrolysate was studied,and the effect of the ratio of acetonitrile on the enrichment of phosphopeptide in the loading conditions was studied.At the same time,the selectivity of the adsorbent to the standard mixed protein sample was investigated.Finally,the adsorbents were used to selectively enrich phosphorylated proteins from actual samples demonstrating the good selectivity and practicality of the adsorbent materials.