CN103667241A - Hair-like hydrophilic polymer hybridization magnetic nanoparticle immobilized enzyme and preparation method thereof - Google Patents
Hair-like hydrophilic polymer hybridization magnetic nanoparticle immobilized enzyme and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a hair-like hydrophilic polymer hybridization magnetic nanoparticle immobilized enzyme and a preparation method thereof. The preparation method of the hair-like hydrophilic polymer hybridization magnetic nanoparticle mmobilized enzyme comprises the following steps of carrying out in situ polymerization on the surface of a magnetic particle through an SI-ATRP (Surface-Initiated Atom Transfer Radical Polymerization) method to generate a polymethacrylamide glucose polymer chain, and generating an aldehyde group by utilizing an oxidative ring-opening polymer lateral chain glucose; then carrying out protease immobilization by carrying out covalent coupling on the aldehyde group and the N-end amido of protease. According to the invention, an enzymatic hydrolysate and the immobilized enzyme can be separated through an external magnetic field; the immobilized enzyme and a protein sample can complete protein enzymolysis by only hatching for 1-2 minutes under the condition of 37 DEG C after being mixed, so that the enzymolysis time greatly shortened; the problem of nonspecific adsorption of the traditional hydrophobic immobilized enzyme material on protein and the enzymatic hydrolysate thereof is solved, so that the sample recovery rate is enhanced. Finally, the immobilized enzyme disclosed by the invention is easy, convenient and fast to use and good in stability, can be repeatedly used, and has good application prospect in large-scale protein group sample analysis.
Description
Technical field
The present invention relates to a kind of hair-like hydrophilic polymer hydridization magnetic-particle immobilization proteinase and preparation method thereof.
Background technology
As the priority research areas of genome times afterwards comprehensively, proteomics research can not only be provided fundamental basis for illustrating of life mechanics, also can be for the early diagnosis of many various diseases, the discovery of medicine target, curative effect judgement and prognosis provide important evidence.From calendar year 2001, by " science " magazine, be chosen as behind the six large hot research fields of 21 century, proteomics research has been subject to scientist's extensive concern.At present, the protein group based on " shotgun " identifies that strategy is to be most widely used and the qualitative and quantitative study strategy of successful complex proteins group sample.This strategy depends on the locus specificity enzyme digestion reaction of proteolytic enzyme to protein, thereby is the polypeptide more compatible with Mass Spectrometric Identification by protein digestion, by the mass spectroscopy to polypeptide, obtains the quantitative and qualitative analysis information of corresponding protein.Therefore enzymolysis efficiency and the thoroughness of protein are the committed steps in sample preparation process in proteome research, and the reliability of final qualification result and accuracy are had to vital impact.Traditional in-solution digestion method is autotomyed for fear of proteolytic enzyme, often use lower proteolytic enzyme ratio (proteolytic enzyme: substrate=1: 50), therefore incubation time (12-20 hour) that need to be longer, limited analysis throughput and the enzymolysis thoroughness of sample, cannot meet that protein science is extensive, high-throughput is qualitative and the requirement of split hair caccuracy quantitative analysis.
Based on above reason, develop protein digestion technology fast and efficiently, for the analysis of complex proteins group sample, be very necessary.Therefore enzyme immobilization technology has been avoided the problem that in traditional in-solution digestion, ubiquitous enzyme is autotomyed, and spendable enzyme concn has obtained raising, has accelerated the process of enzyme digestion reaction, and has recyclable and the plurality of advantages such as reuse.In numerous Carrier Materials of Immobilized Enzyme, as polymeric film, Monolithic Columns, (Xu in porous material, F.et al.Anal.Chem.2010,82,10045-10051.Spross, J.et al.Anal.Chem.2010,82,1434-1443.Qian, K.et al.Anal.Chem.2009,81,5749-5756.), magnetic nanoparticle is because its larger specific surface area and be easy to realize the characteristics such as magnetic resolution and highlight advantage (Lin, S.et al.J. Proteome Res.2008,7,1297-1307.).But what generally adopt at present directly carries out the fixing method of individual layer enzyme at substrate material surface, makes the enzyme supported quantity of unit mass immobilized enzyme material be subject to the long-pending restriction of substrate material surface, thereby has limited the further raising of enzymolysis efficiency.Meanwhile, conventional silicon or hydrophobic polymer matrix material are difficult to avoid protein and enzymolysis product thereof are produced to non-specific adsorption, cause sample loss, thereby affect the sensitivity of identification of proteins and quantitative accuracy.
Transfer Radical Polymerization (Atom Transfer Radical Polymerization, ATRP) is a kind of controllable free radical polymerization process that Matyjaszewki K. professor group proposed first in nineteen ninety-five.Since more than ten years, obtained the extensive concern of international academic community and industry member.ATRP method has the features such as product polymer architecture controllability is good, narrow molecular weight distribution, suitable monomers scope is wide, reaction conditions requirement is moderate.Surface Atom Transfer Radical Polymerization method (SI-ATRP) is ATRP initiator to be fixed on to a kind of method of original position initiated polymerization thing growth after material surface, has now been widely used in finishing and the functionalization of material.
Summary of the invention
The object of this invention is to provide magnetic nano particle immobilized enzyme of a kind of hair-like hydrophilic polymer hydridization and preparation method thereof.
The magnetic nano particle immobilized enzyme of hair-like hydrophilic polymer hydridization provided by the present invention is to prepare according to the method comprising the steps:
1) synthetic a kind of one end is that silane coupling agent, the other end that can be combined with Silica-coated magnetic nanoparticle is surperficial Atom Transfer Radical Polymerization (SI-ATRP) initiator of atom transfer radical polymerization (ATRP) initiator;
2) make the silicon hydroxyl reaction on the magnetic nanoparticle surface of silane coupling agent in described SI-ATRP initiator and Silica-coated, thereby SI-ATRP initiator is covalently attached to the magnetic nanoparticle surface of described Silica-coated;
3) under monomer and catalyst system exist, in step 2) the magnetic nanoparticle surface Atom Transfer Radical Polymerization reaction of the Silica-coated that obtains, at its surface in situ, generate hair-like polymer chain; Wherein, described monomer is can be for the monomer of atom transfer radical polymerization, and in described monomer, contain can with functional group until immobilized enzyme reaction or in described monomer, contain after chemically modified can with the functional group that treats immobilized enzyme reaction;
4) be divided into following a) or b):
A) step 3), in monomer, contain can with the functional group that treats immobilized enzyme reaction, make step 3) described in functional group on polymer chain with treat immobilized enzyme reaction, enzyme is connected on described polymer chain, by not sealing with the functional group of enzyme reaction on described polymer chain, obtain the magnetic nano particle immobilized enzyme of described polymer hybrid again;
B) step 3), in monomer, contain after chemically modified can with the functional group that treats immobilized enzyme reaction, by step 3) described in functional group on polymer chain carry out chemically modified, make its change into can with the functional group that treats immobilized enzyme reaction, and then with treat immobilized enzyme reaction, enzyme is connected on described polymer chain, by not sealing with the functional group of enzyme reaction on described polymer chain, obtain the magnetic nano particle immobilized enzyme of described polymer hybrid again.
Wherein, SI-ATRP initiator step 1) specifically can be obtained by the amino generation amido linkage that reacts with acylbromide with 2-bromine isobutyl acylbromide (ATRP initiator) by APTES (silane coupling agent).And generate siloxane bond by the silane coupling agent in SI-ATRP initiator and the silicon hydroxyl generation dehydration reaction of having wrapped up the magnetic-particle surface of silicon-dioxide, thereby SI-ATRP initiator is covalently bound to magnetic-particle surface.
Step 2) magnetic nanoparticle described in specifically can be ferroferric oxide magnetic nanoparticle, and its particle diameter can be 20nm, and after Silica-coated, particle diameter is 57.4nm.
Step 3) described in, monomer is preferably hydrophilic monomer, specifically can be: Methacrylamide glucose (GMA-G), acrylic monomer, butylene acids monomer or amylene acids monomer etc.Described GMA-G monomer is to be obtained by the amino reaction after the oxidized generation aldehyde radical of glycidyl methacrylate and on glucosamine.When adopting GMA-G monomer to prepare polymer chain, after having prepared, need the adjacent diol structure in the glucose on hydrophilic polymer side chain to be oxidized to aldehyde radical, then with until immobilized enzyme reaction (when the enzyme being connected is Trypsin, to utilize aldehyde radical to react with N-end and the side chain amino of proteolytic enzyme), enzyme is connected on described polymer chain, more remaining aldehyde radical on polymer chain is sealed with monoethanolamine.
Step 3) catalyst system described in is comprised of catalyzer and part.Described catalyzer is selected from the halogenide of following any one metal: Cu, Mo (IV), Ru, Rh, Fe, Re, Ni, Pd and pb; Preferred cuprous chloride.
Described part be selected from following any one: 1, Isosorbide-5-Nitrae, 7,7-five methyl diethylentriamine, dipyridyl, Tetramethyl Ethylene Diamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl Triethylenetetramine (TETA) and three (N, N mono-dimethyl aminoethyl) amine.The mol ratio of described monomer, catalyzer, part can be 200: (0.5-5): (0.75-7.5).
In step 3) in, the magnetic nanoparticle that monomer add-on connects SI-ATRP initiator with respect to surface is excessive.Take GMA-G monomer as example, and the proportioning of GMA-G monomer and magnetic nanoparticle can be 2mmol: 50mg.
In the present invention, for immobilized enzyme, comprise various proteolytic enzyme, as trypsinase, protein incision enzyme (Glu-C), intracellular protein enzyme (Lys-c) etc.; And Glycosylase, as Peptide N-glycosidase F (PNGase F) etc.
Hydrophilic polymer hydridization magnetic-particle immobilized enzyme provided by the present invention can be used for the rapid enzymolysis of protein.Only need the protein example of sex change and hydrophilic polymer hydridization magnetic-particle immobilized enzyme are dissolved in 50mM bicarbonate of ammonia (pH=8.0) or phosphoric acid salt (pH=7.8) damping fluid and mix, 37 ℃ of water-baths are hatched and can be completed enzymolysis in 1-2 minute.Conventionally use 1-10mg particle immobilized enzyme to realize effective enzymolysis to 100 μ g protein.After enzymolysis completes, can use magnet that magnetic-particle immobilized enzyme is adsorbed on tube wall, and draw the enzymolysis product in supernatant liquid.Simple, fastly realize the separated of immobilized enzyme and enzymolysis product, and immobilized enzyme is reclaimed, reused.General reusable 50 left and right of this hydrophilic polymer hydridization magnetic-particle immobilized enzyme.After each enzymolysis protein matter, use 50mM bicarbonate of ammonia (pH=8.0) that immobilized enzyme is cleaned 3 times, remove residual peptide section.Be kept at afterwards in 4 ℃ of refrigerators.
Hydrophilic polymer hydridization magnetic-particle immobilized enzyme provided by the present invention also can be used for efficient, the complete enzymolysis of protein.The bovine serum albumin (BSA) of take is sample, and the peptide section identifying in immobilized enzyme enzymolysis product can reach 93% to the sequential covering rate of BSA; After complex proteins sample enzymolysis, adopt SDS-PAGE to enzymolysis product analysis, without obvious protein residue.
Single protein digestion reaction: take bovine serum albumin as example.By bovine serum albumin (BSA), be dissolved in 50mM bicarbonate of ammonia (pH=8.0) or phosphate buffered saline buffer (pH=7.8), concentration is 0.5mg/ml.Add after dithiothreitol (DTT) (DTT, 10mM), then be placed in boiling water heat denatured 10min, add after cooling iodo-acid amide (IAA, 50mM), 1h is placed in dark place.Hydrophilic polymer hydridization magnetic-particle immobilizing trypsinase (ammonium bicarbonate buffers) or immobilized protein restriction endonuclease Glu-C (phosphate buffered saline buffer) added in the protein example solution of sex change and mix, 37 ℃ of water-baths were hatched after 1 minute, use magnet that magnetic-particle immobilized enzyme is adsorbed on tube wall, and draw the enzymolysis product in supernatant liquid.
Complex proteins sample enzyme digestion reaction: the Tengchong thermophile bacteria whole protein of take is example: add DTT (10mM) in Tengchong thermophile bacteria whole protein sample, in 37 ℃ of water-baths, reduce after 4h, be cooled to room temperature, add IAA (50mM), dark place is placed 1h and is carried out alkylation.The protein example of sex change is dissolved in 50mM bicarbonate of ammonia (pH=8.0) and is mixed with hydrophilic polymer hydridization magnetic-particle immobilizing trypsinase, 37 ℃ of water-baths were hatched after 2 minutes, use magnet that magnetic-particle immobilized enzyme is adsorbed on tube wall, and draw the enzymolysis product in supernatant liquid.
Enzymolysis product is put on ground substance assistant laser desorption ionization time-of-flight mass spectrometer target, added matrix to carry out mass spectroscopy; Or use chromatography-electrospray-ionization/mass spectrometry coupling to analyze enzymolysis product.
Take GMA-G as polymerization single polymerization monomer, prepare the magnetic nano particle immobilized trypsinase of hair-like hydrophilic polymer hydridization and immobilization protein incision enzyme (Glu-C) for example, the principle of the inventive method is illustrated as follows:
As shown in Figure 1, first silane coupling agent and ATRP initiator are synthesized to SI-ATRP initiator by amido linkage covalent coupling.By silane coupling agent and silicon hydroxyl reaction, SI-ATRP initiator is fixed to the magnetic nanoparticle surface of Silica-coated afterwards.The glycidyl acrylate class reagent of take afterwards reacts the hydrophilic reagent GMA-G generating with glucosamine be monomer, cuprous chloride is catalyzer, 1, 1, 4, 7, 7-five methyl diethylentriamine is part (according to the mol ratio of 200: 1: 1.5), mix with the magnetic nanoparticle of having fixed initiator, cause SI-ATRP reaction, at magnetic-particle surface in situ, generate a large amount of hair-like PMAm glucose glycopolymers chains, afterwards the o-dihydroxy group on polymer lateral chain glucose is reacted by sodium periodate oxidation, be converted into aldehyde radical, and utilize two not alkali reaction by the N-end of introduced aldehyde radical and proteolytic enzyme or side chain is amino reacts, be fixed on hydrophilic polymer side chain.
During use, directly protein is mixed with hydrophilic polymer hydridization magnetic-particle immobilized enzyme, in 37 ℃ of water-baths, hatch 1-2 minute, can complete enzymolysis.Enzymolysis product in take-off pipe is put on ground substance assistant laser desorption ionization time-of-flight mass spectrometer target afterwards, carries out mass spectroscopy.Also can use chromatogram automatic sample handling system, carry out chromatography-electrospray-ionization/mass spectrometry combination analysis.
Technical scheme provided by the invention has the following advantages:
1, the magnetic nanoparticle surface of Silica-coated is modified by the standby hair-like hydrophilic polymer chains of SI-ATRP legal system, polymer lateral chain is with a large amount of aldehyde radical functional group, and noncrosslinking hair-like hydrophilic polymer chains also can support 3 D stereo, multilayer proteolytic enzyme to fix.Therefore, compare the supported quantity of the proteolytic enzyme that this hair-like hydrophilic polymer hydridization magnetic-particle immobilized enzyme can obviously improve with the immobilized enzyme that adopts conventional monolayers replace mode to prepare.
2, with traditional immobilized enzyme, proteolytic enzyme being fixed on to hard substrate material surface compares, proteolytic enzyme in this hydrophilic polymer hydridization magnetic-particle immobilized enzyme is fixed on soft polymer chain, there is larger degree of freedom, be conducive to fully contacting of enzyme and protein substrate, thereby promote the carrying out of enzymolysis.
3, need within 12-20 hour, just can complete and compare with traditional in-solution digestion, this hydrophilic polymer hydridization magnetic-particle immobilized enzyme completes protein digestion required time extremely short (1-2 minute), has greatly accelerated sample preparation speed.
4, compare with the immobilized enzyme that uses conventional hydrophobic body material to prepare, this hydrophilic polymer matrix material has significantly reduced the non-specific adsorption to protein and enzymolysis product thereof, thereby has improved the accuracy of sample recovery rate and quantification of protein.
5, this immobilized enzyme is easy to use.After enzymolysis completes, can enzymolysis product is separated with magnetic-particle immobilized enzyme by a step magnetic resolution.
6, this immobilized enzyme has very high enzymolysis efficiency, thoroughness and sample recovery rate, reliability, accuracy and the sensitivity of result when contributing to improve complex proteins group sample and identifying that with " shotgun " strategy carries out qualitative and quantitative analysis, be therefore specially adapted to the Treatment Analysis of complex proteins group sample.
7, enzyme can be repeatedly used.After each use, can be reclaimed by magnetic resolution.Reuse through 50 times, enzymolysis efficiency has no decline.
Accompanying drawing explanation
Fig. 1 is the process flow sheet that the present invention prepares the magnetic nano particle immobilized proteolytic enzyme of hair-like hydrophilic polymer hydridization.
Fig. 2 is fluorescent protein B SA-FITC and the mixed fluorescent microscope photo of Silica-coated magnetic-particle (a) without hydrophilic polymer-modified; With 200 μ LPBS (PH=7.4), clean the fluorescent microscope photo (b) after 5 times; The mixed fluorescent microscope photo of BSA-FITC and hydrophilic polymer hydridization magnetic-particle (c); With 200 μ LPBS (PH=7.4), clean the fluorescent microscope photo (d) of 1 time.
Fig. 3 is the thermogravimetric analysis curve (a) without the magnetic nanoparticle of hydrophilic polymer-modified; The thermogravimetric analysis curve (b) of hydrophilic polymer hydridization magnetic-particle.
Fig. 4: the aminoacid sequence fraction of coverage of the peptide section that BSA standard protein identifies after 12 hours through solution trypsin digestion (allowing 1 leakage to cut site) to BSA.Italics is the aminoacid sequence for identifying partly.
Fig. 5: the aminoacid sequence fraction of coverage of the peptide section that BSA standard protein identifies after 1 minute through hydrophilic polymer hydridization magnetic-particle immobilizing trypsinase enzymolysis (allowing 1 leakage to cut site) to BSA.Boldface letter is the aminoacid sequence for identifying partly.
Fig. 6: the aminoacid sequence fraction of coverage of the peptide section (allowing 1 leakage to cut site) that BSA standard protein identifies after 1 minute through hydrophilic polymer hydridization magnetic-particle immobilization protein incision enzyme Glu-C enzymolysis to BSA.Boldface letter is the aminoacid sequence for identifying partly.
Fig. 7 be Tengchong thermophile bacteria whole protein extract respectively after in-solution digestion and hydrophilic polymer hydridization magnetic-particle immobilized enzyme enzymolysis, use 10%SDS-PAGE (silver dyes) to investigate enzymolysis thoroughness.Swimming lane 1 is protein molecular weight standard (Protein marker), and swimming lane 2 is the Tengchong thermophile bacteria whole protein extract without enzymolysis, and swimming lane 3 is in-solution digestion product, and swimming lane 4 is immobilized enzyme hydrolysis products.In swimming lane 2-4, every swimming lane applied sample amount is 2 μ g.
Embodiment
Below by specific embodiment, method of the present invention is described, but the present invention is not limited thereto.Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.
In following embodiment, the ferroferric oxide magnetic nanoparticle of Silica-coated used prepares by the following method: by 2.31g ferroferric oxide magnetic nanoparticle (purchased from Beijing Deco Dao Jin Science and Technology Ltd., particle diameter is 20nm) add in 40ml ethanol after ultrasonic 60min, in this suspension, add 1.5ml 25% (v/v) ammoniacal liquor, 6ml water, 1.5ml tetraethoxy, in 40 ℃ of water-baths after vigorous stirring 2h, after ultrasonic 60min, with 40ml ethanol rinsing 3 times, Eddy diffusion in 40mL ethanol, 60 ℃ of reflux 12h.Finally gained Silica-coated magnetic nanoparticle is suspended from again in 60mL ethanol to room temperature preservation standby.Grain diameter after Silica-coated is 57.4nm, and silicon dioxide thickness is 18.7nm.
According to the technical process shown in Fig. 1, be prepared.
Synthesizing of 1.1SI-ATRP initiator.Use APTES and 2-bromine isobutyryl bromine reaction synthesize one end for can with the silane coupling agent of Silica-coated magnetic nanoparticle surface bonding, the other end is the SI-ATRP initiator of ATRP initiator.Specific as follows: 8mmol 3-aminopropyl triethoxysilane and 10mmol triethylamine are joined in 12.5ml tetrahydrofuran (THF), after mixing, pass into nitrogen deoxygenation ice bath 30min simultaneously, afterwards 10mmol 2-bromine isobutyl acylbromide is slowly added drop-wise in mixed solution and vigorous stirring 4h (continuing logical nitrogen), finally solution filter final vacuum is dried to initial volume 1/3 to remove tetrahydrofuran (THF) and triethylamine, centrifugal rear removal precipitation, after drying up, nitrogen can obtain yellow thick SI-ATRP initiator, airtight 4 ℃ of preservations after inflated with nitrogen.
Fixing of 1.2SI-ATRP initiator.Utilize the silane coupling agent on SI-ATRP initiator covalently bound with the silicon hydroxyl generation dehydration reaction generation siloxane bond on the magnetic nanoparticle surface of parcel silicon-dioxide, thereby SI-ATRP initiator is fixed on magnetic-particle surface.First by acid-alkali treatment, expose the silicon hydroxyl on Silica-coated magnetic-particle surface, that is: use 0.1M HCl solution soaking magnetic-particle, after 30 minutes by HCl solution removal, it is 7 left and right that water cleans magnetic-particle to pH value of solution, re-use 0.1M NaOH solution soaking magnetic-particle, after 30 minutes, by NaOH solution removal, it is 7 left and right that water cleans magnetic-particle to pH value of solution.Afterwards the ethanolic soln that contains 500 μ M SI-ATRP initiators is mixed with the magnetic-particle after processing, react after 10 hours and remove and remain initiator, and clean magnetic-particle with methanol solution under room temperature, nitrogen dries up.
Synthesizing of 1.3 Methacrylamide glucose monomers.In 674.53 μ l glycidyl methacrylate (GMA), add 0.2M sulfuric acid, be positioned in 50 ℃ of water-baths and heat 4 hours.Add 1.09g sodium periodate to mix, room temperature lucifuge was placed over 2 hours again.Take 0.4 gram of dissolution of sodium hydroxide in 20 ml methanol, add 1.1 grams of glucosamines, be stirred to dissolving.The GMA of oxidation is dropwise added in the DAS continue stirring, stir until be yellow transparent solution, and the solution nitrogen preparing is vapored away to unnecessary methyl alcohol to product be paste, after airtight inflated with nitrogen, place 4 ℃ of preservations.
1.4 cause SI-ATRP reaction, at the magnetic nanoparticle surface in situ generation PMAm glucose polymer chain of parcel silicon-dioxide.Monomer used be one end synthetic in 1.3 with two key the other ends the Methacrylamide glucose with glucose.Monomer, catalyzer (cuprous chloride), part (1, Isosorbide-5-Nitrae, 7,7-five methyl diethylentriamine) are added in methyl alcohol according to mol ratio at 200: 1: 1.5 and supersound process makes its dissolving and mixes.Above-mentioned reaction solution is mixed with the magnetic-particle of having fixed SI-ATRP initiator, adopt freezing-vacuumize-thaw-method of rushing nitrogen removes the oxygen in system, so circulate 3 times, afterwards with sealed membrane sealing, room temperature reaction 24 hours.Reacted rear and cleaned, remove residual reactant with PBS (pH=8.0), obtained hair-like hydrophilic polymer hydridization magnetic nanoparticle.Adopt dynamic light scattering to characterize hair-like hydrophilic polymer hydridization magnetic nanoparticle, grain diameter is 84.4nm, and hair-like hydrophilic polymer layer thickness is 13.5nm.
The aldehyde radical functionalization of the surface hydrophilic polymer lateral chain glucose of the magnetic-particle of 1.5 parcel silicon-dioxide.Concrete grammar is as follows: 10mM sodium periodate is mixed with the hydrophilic polymer hydridization magnetic-particles of 1.4 preparations, room temperature reaction 2 hours.
1.6 carry out proteolytic enzyme on the hydrophilic polymer side chain on magnetic-particle surface fixes.Concrete grammar is as follows, 2mg trypsinase or protein incision enzyme Glu-C are dissolved in to 1ml 50mM ammonium bicarbonate soln (pH=8.0), and add 0.1ml50mg/ml sodium cyanoborohydride solution, after mixing, join 0.2ml concentration and be in the hydrophilic polymer hydridization magnetic-particle solution of aldehyde radical functionalization of 250mg/ml, 4 ℃ of reactions were used magnet that hydrophilic polymer hydridization magnetic-particle is adsorbed in to tube wall after 12 hours, removed supernatant liquor.By this supernatant liquor is calculated to residual enzyme amount in supernatant liquor at the ultraviolet absorption value at 280nm place, thereby show that trypsinase or the supported quantity of protein incision enzyme Glu-C on hydrophilic polymer hydridization magnetic-particle are respectively 247 μ g/mg or 213 μ g/mg.Use afterwards 50mM ammonium bicarbonate soln to clean, remove residual reactant.
The sealing of 1.7 hydrophilic polymer side chain residue aldehyde radicals.Configuration containing 10% (V/V) monoethanolamine phosphate buffered saline buffer PBS (pH=8.0), it is mixed with the hydrophilic polymer hydridization magnetic-particle of having fixed proteolytic enzyme, react 4 hours at 4 ℃.With 50mM ammonium bicarbonate soln, repeatedly clean afterwards, standby after freeze-drying.Obtain the magnetic nano particle immobilized proteolytic enzyme of hair-like hydrophilic polymer hydridization.
As shown in Figure 2, Fig. 2 a is magnetic-particle and the mixed fluorescent microscope photo of fluorescent protein B SA-FITC of parcel silicon-dioxide, and Fig. 2 b is that PBS (PH=7.4) solution cleans fluorescent microscope photo after this magnetic-particle 5 times; Fig. 2 c is the mixed fluorescent microscope photo of hydrophilic polymer hydridization magnetic-particle and BSA-FITC, and Fig. 2 d is that PBS (PH=7.4) solution cleans the fluorescent microscope photo after this magnetic-particle 1 time.As can be seen from Figure, after hydrophilic polymer-modified, on the particle cleaning, substantially do not have fluorescin residual, and the magnetic-particle of parcel silicon-dioxide there are a large amount of fluorescins residual after cleaning.
Thereby hair-like hydrophilic polymer hydridization magnetic nanoparticle causes the polymer layer of mass loss and not heat decomposable magnetic-particle core two portions to form by thermal decomposition.Take respectively 500mg hydrophilic polymer hydridization magnetic nanoparticle and not modified magnetic nanoparticle, carry out thermogravimetric analysis, the results are shown in Figure 3.As can be seen from Figure 3, without polymer-modified magnetic-particle, with the raising of thermal treatment temp, without obvious mass loss, (curve a).And polymer-modified magnetic-particle, raising with thermal treatment temp, show obvious mass loss (approximately 24.17%), prove that it is highly effective by SI-ATRP method, at magnetic-particle in situ Polymerization, generating Methacrylamide glucose polymer.
Take appropriate bovine serum albumin (BSA) standard protein (its sequence is as shown in sequence 1), be dissolved in 50mM ammonium bicarbonate soln in (pH=8.0), final concentration is 2 μ g/ μ l.Adding 10mM mercaptoethanol (DTT), is 5: 1 by the final concentration molar ratio of IAA and DTT, adds IAA solution, and dark place is placed 1 hour by protein denaturation.The protein soln 50 μ l (concentration 2 μ g/ μ l) that get sex change mix with 10mg hydrophilic polymer hydridization magnetic-particle immobilizing trypsinase, be placed in 37 ℃ of water-baths and react after 1 minute and use magnet that magnetic-particle immobilized enzyme is adsorbed in to tube wall, and the protease hydrolysis products in supernatant is taken out.Get the protein soln of the sex change of same amount, according to conventional enzymatic hydrolysis condition, 1: 50 in mass ratio (trypsinase: protein substrate) add trypsinase, be placed in 37 ℃ of water-baths and hatch that to add mass concentration after 12 hours be that 0.1%TFA (trifluoroacetic acid) is by trypsinase deactivation.Get respectively immobilized enzyme enzymolysis and conventional soln enzymolysis product point on ground substance assistant laser desorption ionization time-of-flight mass spectrometer target and natural air drying, CHCA matrix (5mg/ml again, be dissolved in 50% acetonitrile, in 0.1%TFA), carry out mass spectroscopy after to be crystallized.The data obtained file is submitted to respectively online MASCOT and is searched library software and carry out peptide mass fingerprinting spectrum analysis.Analytical results shows, in a leakage of permission, cut under the condition in site, the peptide section identifying from solution trypsin digestion product has only covered 66% (Fig. 4) of BSA aminoacid sequence, and by immobilizing trypsinase enzymolysis gained BSA sequential covering rate up to 93% (seeing Fig. 5).Adopt the hair-like hydrophilic polymer hydridization magnetic-particle immobilizing trypsinase of embodiment 1 preparation to carry out enzymolysis to BSA, reuse after 50 times, enzymolysis efficiency still reaches more than 90%.
Take appropriate bovine serum albumin (BSA) standard protein, be dissolved in 50mM phosphate buffered saline buffer (pH=7.8), final concentration is 2 μ g/ μ l.Adding 10mM mercaptoethanol (DTT), is 5: 1 by the final concentration molar ratio of IAA and DTT, adds IAA solution, and dark place is placed 1 hour by protein denaturation.The protein soln 50 μ l (concentration 2 μ g/ μ l) that get sex change mix with 10mg hydrophilic polymer hydridization magnetic-particle immobilization protein incision enzyme Glu-C, be placed in 37 ℃ of water-baths and react after 1 minute and use magnet that magnetic-particle immobilized enzyme is adsorbed in to tube wall, and the protease hydrolysis products in supernatant is taken out.Get immobilized enzyme enzymolysis product point on ground substance assistant laser desorption ionization time-of-flight mass spectrometer target and natural air drying, the upper CHCA matrix (5mg/ml, is dissolved in 50% acetonitrile, in 0.1%TFA) of point, carry out mass spectroscopy after to be crystallized.The data obtained file is submitted to respectively online MASCOT and is searched library software and carry out peptide mass fingerprinting spectrum analysis.Analytical results shows, allowing a leakage to cut under the condition in site, by immobilization protein incision enzyme Glu-C enzymolysis gained BSA sequential covering rate up to 92% (seeing Fig. 6).Adopt the hair-like hydrophilic polymer hydridization magnetic-particle immobilization protein incision enzyme Glu-C of embodiment 1 preparation to carry out enzymolysis to BSA, reuse after 50 times, enzymolysis efficiency still reaches more than 90%.
Tengchong thermophile bacteria whole protein extracts
Configuration whole protein extracting solution: 50mM Tris-HCl (pH=8.0), 8M urea, 2mM EDTA." cocktail " formula proteinase inhibitor (Roche Germany) that adds 10 μ l to 500 μ l extracting solutions, joins in the test tube that Tengchong thermophile bacteria is housed ultrasonication cell after mixing.Centrifugal 20 minutes of 20000g at 4 ℃, removes not smudge cells and fragment afterwards, and solution is Tengchong thermophile bacteria whole protein.The cold acetone solution (20 ℃ of precoolings) that adds afterwards 4-5 times of volume in protein soln, after-20 ℃ of placements are greater than 2 hours, 4 ℃ of 12000g centrifugal 10 minutes, carefully draw supernatant, retain precipitation.Precipitation is positioned in stink cupboard, makes to remain acetone and fully volatilize.The protein that acetone precipitation is obtained is again dissolved in 8M urea buffer solution and adopts Bradford method to measure protein concn.
Tengchong thermophile bacteria whole protein immobilized enzyme enzyme is cut
Configuration 2mg/ml Tengchong thermophile bacteria whole protein solution, adds DTT, in 37 ℃ of water-baths, reduces 4 hours, adds afterwards IAA to place and within 1 hour, carry out alkylation in dark place.In the protein soln of handling well, add 50mM ammonium bicarbonate soln, after mixing, be divided into two parts, portion adds 10 μ l hydrophilic polymer hydridization magnetic-particle immobilizing trypsinases of embodiment 1 preparation, in 37 ℃ of water-baths, hatch after 2 minutes, use magnet that magnetic-particle immobilized enzyme is adsorbed in to tube wall, draw the enzymolysis product in supernatant.Another is part according to conventional soln enzymatic hydrolysis condition, adds (the trypsinase that mass ratio is 1: 50: protein substrate) trypsinase, is placed in 37 ℃ of water-baths and hatches after 16 hours and add 0.1%TFA by trypsinase deactivation.Get respectively immobilized enzyme enzymolysis and conventional soln enzymolysis product and use chromatography-electrospray-ionization/mass spectrometry coupling to identify, and the data that evaluation is obtained are used MASCOT software to search storehouse.Use conventional soln enzymolysis, single liquid-matter is used in conjunction analysis, from Tengchong thermophile bacteria whole protein extracting solution, identifies altogether 439 protein; And use immobilization enzymolysis, under same evaluation condition, identify altogether 468 protein.Immobilized enzyme enzymolysis has improved the evaluation quantity of protein when significantly shortening enzymolysis required time.Use 10% SDS-PAGE to investigate discovery to the residual protein of the not enzymolysis in immobilization enzymolysis and in-solution digestion product: in in-solution digestion product, to have many bands corresponding to residual protein to occur (Fig. 7, swimming lane 3), and in the product of immobilization enzymolysis, substantially can't see the band that residual protein is corresponding (Fig. 7, swimming lane 4).These results suggest that hydrophilic polymer hydridization magnetic-particle immobilized enzyme can be successfully applied to quick, the efficient enzymolysis of complex proteins group sample.
Claims (10)
1. a method of preparing the magnetic nano particle immobilized enzyme of polymer hybrid, comprises the steps:
1) synthetic a kind of one end, for the surperficial Atom Transfer Radical Polymerization initiator that silane coupling agent, the other end that can be combined with Silica-coated magnetic nanoparticle are atom transfer radical polymerization initiator, is designated as SI-ATRP initiator;
2) make the silicon hydroxyl reaction on the magnetic nanoparticle surface of silane coupling agent in described SI-ATRP initiator and Silica-coated, thereby SI-ATRP initiator is covalently attached to the magnetic nanoparticle surface of described Silica-coated;
3) under monomer and catalyst system exist, in step 2) the magnetic nanoparticle surface Atom Transfer Radical Polymerization reaction of the Silica-coated that obtains, at its surface in situ, generate hair-like polymer chain; Wherein, described monomer is can be for the monomer of atom transfer radical polymerization, and in described monomer, contain can with functional group until immobilized enzyme reaction or in described monomer, contain after chemically modified can with the functional group that treats immobilized enzyme reaction;
4) be divided into following a) or b):
A) step 3), in monomer, contain can with the functional group that treats immobilized enzyme reaction, make step 3) described in functional group on polymer chain with treat immobilized enzyme reaction, enzyme is connected on described polymer chain, by not sealing with the functional group of enzyme reaction on described polymer chain, obtain the magnetic nano particle immobilized enzyme of described polymer hybrid again;
B) step 3), in monomer, contain after chemically modified can with the functional group that treats immobilized enzyme reaction, by step 3) described in functional group on polymer chain carry out chemically modified, make its change into can with the functional group that treats immobilized enzyme reaction, and then with treat immobilized enzyme reaction, enzyme is connected on described polymer chain, by not sealing with the functional group of enzyme reaction on described polymer chain, obtain the magnetic nano particle immobilized enzyme of described polymer hybrid again.
2. method according to claim 1, is characterized in that: step 1) described in SI-ATRP initiator by APTES and 2-bromine isobutyl acylbromide, by the amino generation amido linkage that reacts with acylbromide, obtained.
3. method according to claim 1 and 2, is characterized in that: step 3) described in monomer be hydrophilic monomer, be preferably Methacrylamide glucose, acrylic monomer, butylene acids monomer or amylene acids monomer.
4. method according to claim 3, it is characterized in that: be described step 4): by step 3) described in adjacent diol structure oxidation in glucose on polymer chain generate aldehyde radical, then with treat immobilized enzyme reaction, enzyme is connected on described polymer chain, more remaining aldehyde radical on described polymer chain is sealed with monoethanolamine.
5. according to the method described in any one in claim 1-4, it is characterized in that: step 3) described in catalyst system by catalyzer and part, formed; Described catalyzer is selected from the halogenide of following any one metal: Cu, Mo (IV), Ru, Rh, Fe, Re, Ni, Pd and pb, preferably cuprous chloride;
Described part be selected from following any one: 1, Isosorbide-5-Nitrae, 7,7-five methyl diethylentriamine, dipyridyl, Tetramethyl Ethylene Diamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl Triethylenetetramine (TETA) and three (N, N mono-dimethyl aminoethyl) amine; The mol ratio of described monomer, catalyzer, part is 200: (0.5-5): (0.75-7.5).
6. according to the method described in any one in claim 1-5, it is characterized in that: described enzyme is proteolytic enzyme or Glycosylase; Described proteolytic enzyme is specially trypsinase, protein incision enzyme or intracellular protein enzyme; Described Glycosylase is specially Peptide N-glycosidase F.
7. the magnetic nano particle immobilized enzyme of polymer hybrid that in claim 1-6, described in any one, method prepares.
8. the application of the magnetic nano particle immobilized enzyme of polymer hybrid claimed in claim 7 in protein rapid enzymolysis.
9. a rapid enzymolysis method of protein, comprise the steps: that the protein example of sex change and polymer hybrid magnetic-particle immobilized enzyme are dissolved in pH=8.0,25-100mM ammonium bicarbonate soln or the phosphate buffered saline buffer of pH=7.8 mixing, 25-50 ℃ of water-bath hatched and within 1-2 minute, completed enzymolysis.
10. method according to claim 9, is characterized in that: after described method also comprises that enzymolysis completes, use magnet that described polymer hybrid magnetic-particle immobilized enzyme is adsorbed on tube wall, and draw the enzymolysis product in supernatant liquid.
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| CN105039302A (en) * | 2015-09-08 | 2015-11-11 | 北京蛋白质组研究中心 | Immobilized glycosidase reagent and preparing method thereof |
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| CN104524582A (en) * | 2014-09-24 | 2015-04-22 | 北京化工大学 | Method for constructing multifunctional gene therapy carrier and drug carrier based on silicon monoxide nanoparticles |
| CN104524582B (en) * | 2014-09-24 | 2017-10-10 | 北京化工大学 | It is a kind of that multi-functional gene therapy vector and the method for pharmaceutical carrier are built based on silica nano particle |
| CN105039302A (en) * | 2015-09-08 | 2015-11-11 | 北京蛋白质组研究中心 | Immobilized glycosidase reagent and preparing method thereof |
| CN112679701A (en) * | 2020-12-28 | 2021-04-20 | 重庆宸安生物制药有限公司 | Immobilized lysine endopeptidase and preparation method and application thereof |
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