| Proteomics deals with the large-scale determination of cell and tissue function at protein level.The “shotgun” strategy has been widely used in the field of proteomics research.As a basic and critical step in “shotgun” strategy,the protein enzymatic digestion could directly influence the protein identification.In order to overcome the problems such as contamination by protease,low-efficiency that existed in conventional in-solution proteolysis,enzyme immobilization technology has been taken to improve the performance of free enzyme.However,some new disadvantages like the nonspecific adsorption of proteins and uncompleted release of peptides would come out when using traditional enzyme-immobilized system.To cater to the specific requirement for sample handling of proteomics research,a novel enzyme-immobilized microspheres system was developed.The research including preparation of dual stimuli-responsive microspheres,structure control of the microspheres,the relationship between structures and properties of microspheres,and the application of enzyme-immobilized microspheres in proteomics.The main contents and conclusions are as follows:1.The thermal and pH dual-responsive poly(N-isopropylacrylamide)/poly(acrylic acid)microspheres(PPS)with particle size of 6.5 ?m were successfully synthesized by premix membrane emulsification combined with one-step polymerization.The morphologies of microspheres were characterized by SEM and a laser particle size analyzer.The formation of semi-interpenetrating network structure was confirmed by FT-IR,DSC and TEM.The stimuli-response test demonstrated that the microspheres could respond independently to both pH and temperature.Compared to PNIPAM microspheres,the PPS microspheres showed better dispersibility and stability,which made them ideal for further use in the field of bioengineering.2.To realize the controlling of structures of the microspheres,a series of microspheres were prepared by changing the compositions in water phase,and the relationship between structures and properties of microspheres was investigated.The phase separation behaviors of microspheres during formation process were also analyzed by measuring the variation of transmittance and the gelling time of water phase,and the mechanism of phase separation was analyzed.It was found that the wrinkles on the surface of microspheres and the pores inside microspheres were smaller with higher PAAc content,while the macroporous structures were unchanged.The size of external pores decreased with the increasing AAc content,and the structures of microspheres changed from homogenous semi-interpenetrating network structure to core-shell semi-interpenetrating network structure.The MBA crosslinker largely influenced the structures and properties of microspheres.As the amount of MBA increased from 2.5 to 15 wt%,the structures of microspheres changed from loose structure to homogenous semi-interpenetrating network structure,and further converted to core-shell semi-interpenetrating network structure.The stimuli-response measurement demonstrated that changing PAAc amount hardly impacted the thermal sensitivity and pH sensitivity of microspheres,while increasing AAc or MBA content could enhance the pH sensitivity but weaken the thermal sensitivity.Based on these analyses,the models of semi-interpenetrating network structure in microspheres with different MBA content were proposed.3.The enzyme-immobilized microspheres systems consisting of trypsin and microspheres were prepared by covalent attachment.P(NIPAM-co-AAc)(PcA)microspheres were selected as the model carrier,and the immobilization process was investigated to establish the optimum conditions.Under the optimum parameters for immobilization,enzyme-immobilized microspheres systems were fabricated using PNIPAM/PAAc semi-IPN microspheres(PPS)and P(NIPAM-co-AAc)/PAAc semi-IPN microspheres microspheres(PcPS)as carriers,and the relevant enzymatic properties and stimuli-response behaviors of the systems were studied.The results showed that all the microspheres with high protein loading maintained high enzymatic activity and desirable stimuli-responsive property.Meanwhile,the suitable catalytic temperatures of immobilized enzyme were shifted to the temperatures near LCST values of different microspheres.Because the microspheres were anionic carriers,the suitable catalytic pH of immobilized enzyme shifted slightly to alkaline region.Over a temperature range 25-45 °C,the maximum activity(Vmax)of immobilized enzyme was higher than that of free enzyme.Additionally,the immobilized enzyme possessed much higher thermal stability and storage stability than free enzyme.4.Having recognized the properties of the enzyme-immobilized microspheres,we further expanded their application in protein digestion.The performance of immobilized enzyme was evaluated with three model proteins,i.e.,cytochrome C(Cyt-C),myoglobin(Myo)and bovine serum albumin(BSA)by examining protein sequence coverage,number of peptides with MALDT-TOF and LC-MS MS.The results demonstrated that enzyme-immobilized microspheres could digest Cyt-C with high efficiency,and the digestion time was shortened to 5 min.Three enzyme-immobilized microspheres exhibited protein adsorption ability at temperature higher than LCST,and the resultant peptides could be released completely at temperature lower than LCST.Moreover,in complex protein sample digestion,low molecular weight proteins were preferentially digested using Try-PPS and Try-PcPS.These results made the enzyme-immobilized microspheres ideal for further use in digestion of complex protein mixture.5.The HepG2 cell extract was digested by enzyme-immobilized microspheres under different digestion conditions,and the proteome analysis was performed.It was found that using infrared radiation or changing pH values to acidic region of reaction system could improve the efficiency of digestion.In 30 min digestion under infrared radiation,the number of identified proteins for digest of HepG2 cell extract were 116.The physicochemical characteristics of the identified proteins were investigated.The results showed that(1)more than 60% proteins were smaller than 40 k Da.(2)p I values of most identified proteins were between 4.5 and 7.5,which indicated that the content of extremely acidic and alkali proteins was low.(3)The grand average hydrophobicity(GRAVY)values of most proteins were negative,which meant that the majority of identified proteins were hydrophobic proteins.The cellular location and function of the identified proteins were elucidated by the gene ontology(GO)component and function.The results showed that a large proportion of identified proteins had catalytic activity,and they participated in metabolic process,cell transport,cellular component movement,cell differentiation,cell proliferation and defense response,etc.These results help ones to realize that hepatocytes are of vital importance in metabolism and play a pivotal role in human homeostasis.In this thesis,the micro-sized hydrogel microspheres were prepared by premix membrane emulsification combined with one-step polymerization and a new formulation for enzyme-immobilized microspheres system was achieved.Further,we proposed a new method and mechanism for highly efficient protein digestion,which would advance the application of “smart” thermo-sensitive hydrogel microspheres in the field of proteomics research.Besides,we believe that these original results can provide a foundation for further studies. |
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