Preparation Of High-Performance Magnetic Composite Microspheres And Their Application In Selective Enrichment Of Low Abundant Phosphopeptides And Glycopeptides

Due to the integrated functions of the unique magnetic responsiveness supported by the magnetic core and the desired function offered by the functional shell, multifunctional magnetic composite microspheres have broad application prospect in the biomedical field including bioseparation, medical diagnosis, magnetically targeted drug delivery and so on. Enhancing the properties of magnetic composite microspheres, such as enhancing the magnetic responsiveness and surface functional group density, will help to improve their pracitical application effect. Selective isolation of target proteins or peptides is essential for the successful investigation of proteome or peptidome. With the aim to address the difficulties in selective enrichment of glycopeptides, selective enrichment of phosphopeptides as well as selective extraction of phosphopeptidome, this thesis has focused on preparing several kinds of high-performance magnetic composite microspheres and developping a series of new techniques. The main results are summarized as following:(1) A facile and efficient route for the preparation of MSP@PMAA composite microspheres was developped. Firstly, magnetic supraparticles (MSPs) stabilized by citrate were synthesized through a modified solvothermal reaction. Secondly, the MSPs were modified with MPS to form active C=C bond on the surface of MSPs. Finally, a robust layer of PMAA was coated on the MSP@MPS core by distillation-precipitation polymerization of MAA (monomer) and MBA (crosslinker) to form monodisperse MSP@PMAA core/shell composite microspheres without the aid of any intermediate layer. All kinds of characterization methods indicated that the MSP@PMAA microspheres possess well-defined core/shell structure, abundant surface carboxylic groups, high saturation magnetization and great pH sensitivity. The experimental results proved that the thickness and degree of crosslinking of PMAA shell could be well-controlled by adjusting the reaction concentration and the feeding ratio of MBA to MAA respectively. Moreover, we also demonstrated that other magnetic polymeric microspheres with similar structure, such as MSP@PAA, MSP@PAM and MSP@PNIPAM, could be synthesized by this approach.(2) Based on the MSP@PMAA microspheres, Ag nanoparticles were deposited onto the surface of MSP@PMAA by a simple in-situ reduction of Ag+ in the presence of butylamine with AgNO3 as the precursor. Taking advantages of the reversible multivalent interaction of glycans with Ag nanoparticles, the MSP@PMAA@Ag-NPs microspheres possess remarkable selectivity for glycopeptides even at a low molar ratio of glycoprotein/non-glycoprotein (1:100) with a very rapid enrichment speed (only need 1 min) and short operation time (only need less than 10 min) using magnetic separation. Applying this approach,463 nonredundant peptides were identified, among which,245 nonredundant peptides were identified as glycopeptides. Within the 245 nonredundant glycopeptides,127 unique glycopeptides are mapped to 51 different glycoproteins.(3) Based on the MSP@PMAA microspheres, MSP@PMAA@PEGMP core/shell/shell composite microspheres were prepared via two-step distillation-precipitation polymerization by utilizing the strong hydrogen bonding interaction between the surface carboxyl groups and the phosphate groups of EGMP. Then Ti4+ was immobilized onto the surface of MSP@PMAA@PEGMP by the coordination reaction between Ti4+ and the phosphate groups of PEGMP via metal(IV) phosphate chemistry. Taking advantage of the pure phosphate-Ti4+ interface and high Ti4+ loading amount, the MSP@PMAA@PEGMP-Ti4+ composite microspheres possess remarkable selectivity, extreme sensitivity, large enrichment capacity and excellent recovery toward enrichment of phosphopeptides. Applying this approach,10 and 4 unique phosphopeptides were enriched from the digestion of drinking milk and human serum respectively.(4) Based on the MSP@PMAA microspheres, large amount of the generated hydrolyzate Zr(OH)4 was firmly fixed onto the surface of the crosslinked polymethylacrylic acid matrix via strong hydrogen bonding interaction between Zr(OH)4 and the carboxyl groups. Then a calcination process was adopted to convert the Zr(OH)4 into a continuous ZrO2 shell and simultaneously make the ZrO2 shell crystallized. At the same time, the polymer matrix could be selectively removed to form yolk-shell structure. By taking advantage of the highly pure and crystalline ZiO2 surface, high magnetic susceptibility as well as the special yolk-shell structure, the yolk-shell MSP@ZrO2 exhibited high specificity and high sensitivity in selective enrichment of phosphopeptides. Applying this approach, a total of 33 unique phosphopeptides containing 49 phosphorylation sites mapped to 33 different phosphoproteins were identified from 1 mL of human saliva. Moreover, the synthetic method is versatile and can be used to produce yolk-shell MSP@ZrO2-TiO2.(5) Based on the MSPs stabilized by citrate, a compact TiO2 layer was directly deposited on the surface of MSPs using a sol-gel method with NH3@H2O as catalyst and TBOT as precursor. Then the obtained MSP@TiO2 was subjected to a hydrothermal crystallization process in a mixed solvent containing ethanol and deionized water, which led to the formation of a crystalline and mesoporous TiO2 shell. The reaction condition of hydrothermal crystallization was rigorously investigated to find out the optimal condition. The as-synthesized tailor-made MSP@mTiO2 microspheres possess high magnetic susceptibility, high crystallinity, high specific surface area and good dispersability in solvent. Taking advantage of these unique properties, the MSP@mTiO2 showed remarkable selectivity, extreme sensitivity, excellent enrichment capacity and high recovery toward enrichment of phosphopeptides. The reaction time of the hydrothermal crystallization could be further reduced to only 10 min by using microwave volumetric heating, which will help to greatly decrease the energy consumption. In addition, comparing to conventional hydrothermal crystallization, the product prepared with the aid of microwave irradiation has a higher specific area and a smaller average pore diameter.(6) Based on the MSP@TiO2 microspheres, a hydrothermal process at 80℃ was adopted to prepare the custom-made MSP@mTiO2-80 endowed with size-exclusive effect. Thanks to the ultrahigh specific surface area, high TiO2 content and high magnetic susceptibility, the MSP@mTiO2-80 showed remarkable selectivity, extreme sensitivity, exceptional enrichment capacity and excellent recovery toward enrichment of phosphopeptides. In addition, the appropriate pore diameter and the very narrow pore-size distribution make the MSP@mTiO2-80 possess excellent size-exclusion capability against proteins including both nonphosphoproteins and phosphoproteins. Applying this approach, we successfully identified 35 phosphopeptidome in snake venom, which accounted 75% of the total identified peptides.(7) Monodisperse spherical titania (TiO2) microspheres with an average diameter of about 400 nm were firstly synthesized via controlled hydrolysis of TBOT in a mixed solvent. Then the as-prepared TiO2 microspheres were successively coated with a non-crosslinked PMAA interim layer and a crosslinked P(NIPAM-co-MBA) shell by two-step reflux-precipitation polymerization. With the help of the mTiO2 core and crosslinked P(NIPAM-co-MBA) gel network shell, the rattle-type mTiO22@P(NIPAM-co-MBA) exhibited high selectivity and extreme sensitivity toward enrichment of phosphopeptides as well as great size-exclusion effect against both nonphosphoproteins and phosphoproteins. The experiment result suggests that the critical exclusion molecular weight of the P(NIPAM-co-MBA) gel network shell is around 24 kDa. What’s more, magnetic supraparticle core can be conveniently incorporated into the rattle-type mTiO2@P(NIPAM-co-MBA) to enable easy separation by magnetic separation.