Research Of Magnetic Fe3O₄@mTiO₂-aipa Microspheres For Separation Of Phosphoproteins

Protein phosphorylation is a very important post-translational modification,and plays a crucial role in a wide range of biological processes,such as cell differentiation,metabolism and signal transduction.Substantial studies have shown the close relationship between abnormal phosphorylation and many critical diseases,especially the cancers and neurodegenerative disorders.Thus a comprehensive analysis of phosphoproteins is significant for pathological studies and early diagnosis of related diseases.However,because of the low abundance and low ionization efficiency of phosphoproteins,the signal intensity of phosphorylated species is frequently suppressed by the presence of abundant non-modified species and superabundant of salts in mass spectrometry（MS）analysis.Therefore,it is essential to selectively enrich phosphoproteins or phosphopeptides from complicated biosamples before MS-based phosphoproteomics.Currently,there are two complementary lines for phosphoproteomics research:bottom-up strategy based on proteolytic peptides and top-down strategy based on intact phosphoproteins.Compared with the bottom-up approach,the top-down phosphoproteomics is largely hampered by the lack of efficient enrichment materials and methods for intact phosphoproteins.Except for the expensive method of co-immunoprecipitation（Co-IP）,there are few reports about the research on capturing phosphoproteins from biosamples.For this purpose,we incorporated the merits of mesoporous materials,magnetic materials and thiourea-based anion receptors to design a novel organic-inorganic hybrid material for direct separation of phosphoproteins and non-phosphoproteins.The detailed experimental studies and main results are as follows:（1）We designed a 5-（3-propylthioureido）isophthalic acid（AIPA）as a thiourea-based phosphate receptor.Firstly,the clear complexation between AIPA and phosphate group was demonstrated through fluorescence titration experiment,¹H-NMR titration experiment,FT-IR titration experiment and theoretical calculation.The results showed that the binding process was mainly driven by multiple hydrogen bonding interactions between the thiourea or carboxyl groups in AIPA and phosphate group.And the association constant was calculated to be 43100±4900 L·mol^?1.Then,according to the results of quartz crystal microbalance（QCM）adsorption experiments,atomic force microscope measurements and surface contact angle measurements,the interaction between AIPA monolayer and phosphoproteins was further confirmed,and the adsorption quantity could reach 1534 ng?cm^?2.（2）The magnetite Fe₃O₄ nanoclusters were prepared through a solvothermal reaction.Then the Fe₃O₄@mTiO₂ microspheres were synthesized by directly depositing a mesoporous TiO₂ layer（mTiO₂）on the surface of Fe₃O₄ nanoclusters through two steps of sol–gel method and solvothermal reaction.Finally,an AIPA monolayer was grafted onto the surface of Fe₃O₄@mTiO₂ microspheres mediated with silane coupling agent to obtain organic-inorganic hybrid material Fe₃O₄@mTiO₂-AIPA.A series of characterization methods demonstrated that this material with high specific surface area(108.5 m²·g^-1)and suitable pore size（19.75nm）had good dispersability and excellent magnetic property,largely improving adsorption efficiency for phosphoproteins.（3）By adopting the magnetic-solid phase extraction method,Fe₃O₄@mTiO₂-AIPA was successfully utilized to separateα-casein（a typical phosphoprotein）and bovine serum albumin（BSA,a typical non-phosphoprotein）from their mixtures（mass ratio=1:5）.Through adjusting pH and polarity of solutions,the BSA andα-casein were respectively enriched in washing fraction and elution fraction.In summary,this novel magnetic Fe₃O₄@mTiO₂-AIPA material displays a good potential in top-down phosphoproteomics.Furthermore,it will provide a new direction for developing new enrichment and separation materials for phosphoproteins.