CN107586816A - A kind of infrared ray immobilised enzymes coordinated enzymatic hydrolysis method of protein - Google Patents

Abstract

The present invention relates to a kind of infrared ray immobilised enzymes coordinated enzymatic hydrolysis method of protein, and it is to be combined infrared technology with temperature sensitive immobilised enzymes to carry out coordinated enzymatic hydrolysis processing to protein, and any external heat and cooling system are not needed in enzymolysis process.Step of the present invention is simple and convenient to operate, and reaction system is heated up using infrared ray and promotes the exposure of protease cutting site, the acting frequency of restriction enzyme site and enzyme can be effectively increased, so as to significantly improve enzymolysis efficiency；Meanwhile in system temperature-rise period, simultaneously phase in version occurs for temperature sensitive immobilised enzymes microballoon absorption system heat, shows hydrophobic property.Hydrophobic microballoon can effectively keep enzymatic activity, and be enriched with testing protein quality sample by hydrophobic interaction, increase the contact probability of enzyme and protein.This infrared ray acts on immobilised enzymes coordinated enzymatic hydrolysis, and the efficient enzymolysis of protein can be better achieved, and contributes to the high flux of batch protein to digest and identify.

Description

A kind of infrared ray-immobilised enzymes coordinated enzymatic hydrolysis method of protein

Technical field

The present invention relates to belong to biological chemical field, and in particular to a kind of protein digestion method, more particularly to it is a kind of red Outside line-immobilised enzymes coordinated enzymatic hydrolysis method of protein.

Background technology

Proteomics is a subject that the genomics epoch rise behind the door, and it is substantially to base from protein level Cause and cell progress intuitively identify that it is widely used in the identification of cancer and treatment, the prediction of disease etc. on a large scale.Protein Enzymolysis be in proteomics research protein analysis identify committed step.Traditional enzymolysis process is to be based on in-solution digestion Method, typically by free enzyme and testing sample with mass ratio 1:After 50 ratio mixing, 12-24h is reacted at 37 DEG C.This side The problems such as itself enzymolysis, the enzyme that method has longer, the easy generation enzyme of enzymolysis time can not recycle.In order to overcome these to lack Point, scientist have carried out substantial amounts of research to enzyme solution.

At present, the method for improving enzymolysis efficiency mainly uses physico-chemical process and enzyme immobilization technology to enzymolysis process Promoted.Conventional physico-chemical process includes ultrasonic wave, microwave, infrared ray etc..As a kind of important electromagnetic wave, The most commonly used application of infra-red radiation is to provide heating source.Recently, the work of free enzyme enzymolysis protein is aided in using infrared ray Have been reported.For traditional water bath heating, using infrared ray except origin of heat can be provided, it is often more important that Infrared ray can excite the vibration of intramolecular, so as to promote the exposure of protease enzyme site, improve enzymolysis efficiency.It is however, red Outside line is during assistance enzymolysis, and because it persistently excites molecular vibration, the object that can make to be radiated produces heat.Work as temperature When too high, enzyme can be caused to inactivate, influence hydrolysis result.Therefore, when aiding in free enzyme to digest using infrared ray, it is necessary to use outer Source refrigerating plant is controlled to the temperature of reaction system.This considerably increases the complexity of device, and waste the energy.

Enzyme immobilization technology is to fix proteolytic enzyme on a support material, is entered at a certain temperature with protein solution Row enzyme digestion reaction, the technology significantly improve the heat resistance of enzyme by increasing the rigidity of enzyme molecular structure.But enzyme molecule is consolidated After fixed, mobility is deteriorated, while the steric hindrance that carrier is formed between enzyme and testing protein quality sample also limit enzyme and bottom The contact of thing.Therefore, if by testing protein example enrichment around immobilised enzymes, and enzyme can be improved in enzymolysis process With the contact frequency of testing protein quality sample restriction enzyme site, then can high degree accelerate enzyme digestion reaction, shorten enzymolysis time.Consider To the advantage of the excellent effect of infrared ray assistance enzymolysis, and enzyme immobilization technology, if two kinds of technologies can be combined, and not Using cooling device, then a kind of new rapidly and efficiently method can be provided for protein digestion.

The content of the invention

In view of the above-mentioned problems in the prior art, the invention provides a kind of infrared ray-immobilised enzymes coordinated enzymatic hydrolysis Method of protein, the present invention is fixed enzyme vector using Thermo-sensitive microballoon, by infrared technology and temperature sensitive immobilised enzymes phase Coordinated enzymatic hydrolysis processing is carried out with reference to protein, and any external heat and cooling system are not needed in enzymolysis process.This hair Bright step is simple and convenient to operate, and reaction system is heated up using infrared ray and promotes the exposure of protease cutting site, can be had The acting frequency of effect increase restriction enzyme site and enzyme, so as to significantly improve enzymolysis efficiency；Meanwhile in system temperature-rise period, it is temperature sensitive Simultaneously phase in version occurs for immobilised enzymes microballoon absorption system heat, shows hydrophobic property.Hydrophobic microballoon can effectively keep enzyme activity Property, and testing protein quality sample is enriched with by hydrophobic interaction, increase the contact probability of enzyme-to-substrate.This infrared ray is with consolidating Surely change the effect of enzyme coordinated enzymatic hydrolysis, the efficient enzymolysis of protein can be better achieved, contribute to the high-throughput enzyme of batch protein Solution and identification.

To use following technical scheme up to this purpose, the present invention：

The invention provides a kind of method of protein digestion, it includes：Temperature sensitive immobilization is utilized under infrared radiation Enzyme microballoon digests to protein.

The present invention is using Thermo-sensitive microballoon as fixed enzyme vector, and it is micro- that temperature sensitive immobilised enzymes is adjusted by infrared radiation The hydrophobe property of ball, and then control protease solution preocess.

The present invention is combined for protein digestion, i.e. present invention offer using by infrared ray-temperature sensitive immobilised enzymes microballoon On infrared ray-temperature sensitive immobilised enzymes microballoon it is combined the purposes in protein digestion.

The present invention utilizes infrared radiation to adjust the principle of temperature sensitive immobilised enzymes microballoon hydrophilic and hydrophobic：In infrared radiation Under, when temperature is higher than the critical inversion temperature of temperature sensitive immobilised enzymes microballoon, microballoon is in hydrophobicity, can be enriched with testing protein It is digested while quality sample, when temperature is less than the critical inversion temperature of temperature sensitive immobilised enzymes microballoon, stops enzyme Solve and discharge peptide fragment, so as to improve the recall rate of enzymolysis efficiency and sample peptide fragment.

The present invention uses Thermo-sensitive microballoon, and compared to Nano grade carrier, it has the faster rate of settling, without using receiving The harsh separation conditions such as rice carrier system high rotating speed required in separation process, long-time, can be avoided due to for a long time During centrifugation, influence of the centrifuge heating to activity of the immobilized enzyme；Compared to use other carriers, such as magnetic-particle, polymer film, Mesoporous material, silica, graphene oxide composite material etc., it can be realized by temperature to adjust temperature sensitive immobilised enzymes microballoon Hydrophobe property, so as to greatly improve the degree of accuracy of the efficiency of enzymolysis and subsequent protein identification, improve the inspection of sample peptide fragment Extracting rate.

The present invention uses infrared technology, does not need any external heat and cooling system in enzymolysis process, makes full use of Infrared ray is heated up to reaction system and promotes the exposure of protease cutting site, and it has with temperature sensitive solidification both enzyme microballoon Synergistic function, it is achieved thereby that the efficient enzymolysis of protein, contributes to the high flux of batch protein to digest and identify.

According to the present invention, the method for the protein digestion, it comprises the following steps：

(1) protein example to be digested is subjected to thermal denaturation processing；

(2) under infrared radiation, the solution obtained using temperature sensitive immobilised enzymes microballoon to step (1) digest instead Should, when the temperature of reaction solution rises to 45-65 DEG C, close infrared ray；

(3) temperature of reaction solution is down to 5-25 DEG C, enzymolysis reaction, separates temperature sensitive immobilised enzymes microballoon and enzymolysis Reaction mixture, collect enzymolysis product solution.

The enzymolysis product being collected into can be directly subjected to Mass Spectrometric Identification in the present invention, so as to realize the identification of protein, obtained To the bar number of sample peptide fragment.

According to the present invention, power is used as 50-400W, and wavelength is that 2.5-20 μm of infrared ray is radiated.Institute of the present invention Wave number corresponding to the wavelength of infrared facility is 500-4000cm^-1, this wave-number range can include protein molecular just The vibrational wave number of middle chemical bond, such as：Stretching vibration and flexural vibrations, c h bond stretching vibration, the C=O key stretching vibrations of N-H keys With the stretching vibration of C-N keys.These keys constitute the peptide bond in protein molecule, and some of them are the digestions of trypsase Site.Therefore, can cause the vibration of protein peptide bond by infrared ray, improve restriction enzyme site exposure probability, make restriction enzyme site with The acting frequency increase of enzyme, so as to improve enzymolysis efficiency.

According to the present invention, the temperature sensitive immobilised enzymes microballoon used in protein digestion, its carrier material is by N- isopropyls What base acrylamide was formed with acrylic or methacrylic acid by copolyreaction, preferably by NIPA and propylene Acid is formed by copolyreaction.

Used carrier material can be prepared using technology well known in the art in the present invention, such as can be used Emulsion polymerization, radical polymerization or membrane emulsification etc., specifically, such as it can be prepared with the following method.

The first：

1.1 experiment reagent

NIPA, recrystallized 3 times with n-hexane-acetone (volume ratio 50/50) mixed solvent, comonomer For acrylic acid, polymerization inhibitor is removed with 5% NaOH solution, initiator is potassium peroxydisulfate, crosslinking agent N, N '-methylene bisacrylamide Acid amides.

The preparation of 1.2 microballoons

Copolymerization microsphere is prepared using emulsifier-free emulsion polymerization reaction：By monomer NIPA, acrylic acid and crosslinking Agent is put into 250mL four-neck flask after being dissolved in 200mL deionized waters, NIPA, crosslinking agent, deionized water Mass ratio be 100:0.1-1:20000 are passed through nitrogen and carry out mechanical agitation, initiator are added after stable 15min, by temperature 80 DEG C are increased to, continues to react 4-6h.Reaction cools the temperature to room temperature after terminating, and sample is loaded into molecular cut off 20000- In 50000 bag filter at room temperature pure water dialysis 48-72h.Finally, sample is freezed, be sealed at room temperature.

Second：

1.1 experiment reagent

NIPA, recrystallized 3 times with n-hexane-acetone (volume ratio 50/50) mixed solvent, comonomer For methacrylic acid, initiator is azodiisobutyronitrile, and crosslinking agent N, N '-methylene-bisacrylamide, stabilizer is poly- second Alkene pyrrolidone.

The preparation of 1.2 microballoons

Copolymerization microsphere is prepared using dispersion polymerization：0.5-5g stabilizers are added in 50mL water and stirred, by first Base acrylic acid, NIPA, crosslinking agent are added thereto and are passed through nitrogen.NIPA, crosslinking agent, go The mass ratio of ionized water is 1: 0.2-1.0: 80.System temperature is risen to 60 DEG C after 30min, 0.25- is added after stable 15min 0.5g initiators, react 6-12h.Reaction cools the temperature to room temperature after terminating, and sample is loaded into molecular cut off 20000-50000 Bag filter at room temperature pure water dialysis 48-72h.Finally, sample is freezed, be sealed at room temperature.

The third：

1.1 experiment reagent

NIPA, recrystallized 3 times with n-hexane-acetone (volume ratio 50/50) mixed solvent, comonomer Acrylic acid, polymerization inhibitor is removed with 5% NaOH solution, initiator is ammonium persulfate, crosslinking agent N, N '-methylene bisacrylamide acyl Amine.

The preparation of 1.2 microballoons

Microballoon is prepared using precipitation polymerization method：Acrylic acid, NIPA, initiator, crosslinking agent solvent is molten For solution in the 80mL aqueous solution, the concentration of NIPA is 7-10g/L；NIPA, initiation in solution Agent, the mass ratio of crosslinking agent are 10:0.25-1:0.5-1.5；Lead to nitrogen 30min into solution, then in oil bath or water bath condition Under heat the solution to 70 DEG C, react 8-12h；React and reaction solution temperature is down to room temperature after terminating, sample is loaded In molecular cut off 20000-50000 bag filter at room temperature pure water dialysis 48-72h.Finally, sample is freezed, at room temperature It is sealed.

4th kind：

1.1 experiment reagent

NIPA, recrystallized 3 times with n-hexane-acetone (volume ratio 50/50) mixed solvent, copolymerizing agent third Olefin(e) acid, polymerization inhibitor is removed with 5% NaOH solution, initiator is ammonium persulfate, crosslinking agent N, N '-methylene bisacrylamide acyl Amine, accelerator are tetramethylethylenediamine.

1.2 prepare aqueous phase

Acrylic acid, NIPA, initiator, crosslinking agent are added in deionized water, N- isopropyl acrylamides Amine, initiator, crosslinking agent, the mass ratio of deionized water are 10: 0.2-0.8: 0.2-1.5: 500, stir to solid and are completely dissolved, It is configured to aqueous phase；

1.3 prepare oil phase

It is in mass ratio 1: 25 mixing by Span-80, hexamethylene, is stirred at room temperature to Span-80 and is completely dissolved, be configured to oil Phase；

The preparation of 1.4 microballoons

Copolymerization microsphere is prepared using quick membrane emulsification：It is 1 by volume by oil phase and aqueous phase:5-20 is mixed, 140r/ 15min is stirred under min rotating speed and obtains mixed liquor；Under certain nitrogen pressure mixed liquor is pressed through into micropore glass film, and (aperture is 0.5-50 μm), obtain water-in-oil emulsion.Water-in-oil emulsion is added in 150mL four-neck flasks, and stirring at low speed is simultaneously passed through nitrogen.1h Backward emulsion in add accelerator, react 4-6h at 25 DEG C.Reaction stops stirring after terminating, and utilizes acetone and deionized water It is respectively washed microballoon 3 times.To finally clean obtained microballoon be scattered in deionized water preserve it is stand-by.

5th kind：

1.1 experiment reagent

NIPA, recrystallized 3 times with n-hexane-acetone (volume ratio 50/50) mixed solvent, copolymerizing agent third Olefin(e) acid, polymerization inhibitor is removed with 5% NaOH solution, initiator is ammonium persulfate, crosslinking agent N, N '-methylene bisacrylamide acyl Amine, accelerator are tetramethylethylenediamine.

1.2 prepare aqueous phase

Acrylic acid, NIPA, initiator, crosslinking agent are added in deionized water, N- isopropyl acrylamides Amine, initiator, crosslinking agent, the mass ratio of deionized water are 10: 0.6: 1.5: 100, stir to solid and are completely dissolved, are configured to water Phase.

1.3 prepare oil phase

It is 1 in mass ratio by Arlacel-80, decyl alcohol:10 mixing, are stirred at room temperature to Span-80 and are completely dissolved, be configured to oil Phase.

The preparation of 1.4 microballoons

Aqueous phase solution is passed through in the injection-tube (internal diameter is 400 μm) out of micro fluidic device chip；Injection-tube is imported Below oil phase liquid level.During droplet formation, drop can be controlled by controlling speed caused by drop (30-400 μ L/h) Size and its homogeneity.Drop keeps can avoid droplets from unstable of certain distance and merged as far as possible in passage, and aqueous phase is molten Liquid can obtain water-in-oil emulsion after being all passed into oil-phase solution.Water-in-oil emulsion is added in 150mL four-neck flasks, stirring at low speed And it is passed through nitrogen.Accelerator is added in 1h backward emulsion, reacts 4-6h at 25 DEG C.Reaction stops stirring after terminating, and utilizes Acetone and deionized water are respectively washed microballoon 3 times.To finally clean obtained microballoon be scattered in deionized water preserve it is stand-by.

From size uniformity is prepared, the angle of the micron level particle of size tunable is set out, currently preferred preparation method For membrane emulsification.The microballoon that dispersin polymerization, precipitation polymerization and emulsifier-free emulsion polymerization are prepared is mostly Nano grade, and particle diameter is not It is easy to control.Although microflow control technique can prepare the microballoon of uniform particle diameter, its preparation process flux is smaller, yield bottom, seriously Follow-up technique amplification is limited, therefore also not as preferred scheme.

The present invention mass ratio sour with acrylic or methacrylic by adjusting NIPA, can be prepared Hydrogel microsphere with preferable temperature sensitivity and different critical phase transition temperature, such as can be by NIPA 20 are defined to the mass ratio of acrylic or methacrylic acid:1-1:1, preferably 9:1-4:1.In the range of this mass ratio, have Help improve the temperature sensitivity and critical inversion temperature of temperature sensitive microballoon, reach 32-45 DEG C.

According to the present invention, the temperature sensitive immobilised enzymes microballoon used in protein digestion, its immobilised enzymes is immobilization pancreas Protease or Immobilized chymotrgpsin, preferably immobilizing trypsinase.

Used temperature sensitive immobilised enzymes microballoon can be prepared using technology well known in the art in the present invention, specifically Ground, such as can be prepared with the following method.

The first：

Immobilised enzymes is prepared using physisorphtion：Trypsase is dissolved in phosphate buffer solution (buffer solution ion Concentration is 10mmol/L, pH=7.2~7.4), it is configured to 1mg/mL trypsin solution.Take out microballoon and add tryptose In enzyme solutions, the mass ratio of microballoon and trypsase is 2:1~1:2, mixed liquor carries out 12h adsorption reaction at 4 DEG C.Reaction knot Shu Hou, immobilizing trypsinase microballoon is separated with having neither part nor lot in the trypsase of adsorption reaction, it is micro- to take out immobilizing trypsinase Ball is scattered in phosphate buffer solution, is saved backup at 4 DEG C.

Second：

Immobilised enzymes is prepared using chemical covalent fixation.Trypsase is dissolved in phosphate buffer solution (buffer solution Ion concentration is 10mmol/L, pH=7.2~7.4), it is configured to 1mg/mL trypsin solution.On the other hand, microballoon is taken Go out to be dissolved in cushioning liquid, accelerator 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate is dissolved in 1mL PH=4.0 hydrochloric acid solution in, the molal quantity of accelerator is consistent with the molal quantity of the carboxylic group contained by microballoon, by accelerator Solution is mixed with microspheres solution, and the mixed liquor is positioned over to the activated carboxylic reaction that 2h is carried out at 25 DEG C.Reaction is used after terminating Deionized water washing microballoon 3~5 times, removes impurity and is scattered in microballoon in phosphate buffer again.By microspheres solution and pancreas Protein enzyme solution mixes, and the mass ratio of microballoon and trypsase is 2 in solution:1-1:After reacting 12h at 2,4 DEG C, by immobilization Enzyme microballoon separates with having neither part nor lot in the trypsase of reaction.Resulting immobilizing trypsinase microballoon is scattered in phosphate buffer solution In, saved backup at 4 DEG C.

The third：

Immobilised enzymes is prepared using the method for physical absorption and chemical covalent secure bond.

1.1 adsorption reaction

Trypsase is dissolved in phosphate buffer solution (buffer solution ion concentration is 10mmol/L, pH=7.2~ 7.4), it is configured to 1mg/mL trypsin solution.Microballoon is taken out simultaneously to add in trypsin solution, microballoon and trypsase Mass ratio is 1:1-1:2, mixed liquor carries out 2h adsorption reaction at 4 DEG C.

1.2 is covalently fixed

Accelerator is added in 1mL pH=4.0 hydrochloric acid solution, add contain microballoon and tryptose until completely dissolved In the solution of enzyme, the molal quantity of accelerator is consistent with the molal quantity of the carboxylic group contained by microballoon, and the mixed liquor is positioned over into 25 2h covalent bond reaction is carried out at DEG C, then continues to react 10h at 4 DEG C.Reaction terminates, by immobilised enzymes microballoon with not joining Activator, trypsase with reaction separate.Resulting immobilizing trypsinase microballoon is scattered in phosphate buffer solution, and 4 Saved backup at DEG C.

The method that temperature sensitive immobilised enzymes microballoon is prepared in the present invention is preferably physical absorption and chemical covalent secure bond Method.This is due to that physics inhales the immobilised enzymes microballoon that method is prepared, and enzyme is only adsorbed in microsphere surface by electrostatic interaction, Electrostatic interaction is unstable easily to be influenceed by external condition, easily causes coming off for enzyme.And it is prepared into by covalent fixing means The immobilised enzymes arrived, relatively consolidated although being combined between enzyme and microballoon, the transformation of enzyme conformation directly caused with covalently bonded credit union, Cause a large amount of losses of enzymatic activity.And use physical absorption and the method for chemical covalent secure bond to prepare immobilised enzymes microballoon, It can make enzyme and microballoon close to each other under a weaker ion interphase interaction first, and reach the enzyme amount near microballoon Balance, then recycle covalent bond to fix enzyme molecule, be so advantageous to reduce the conformation transition degree of enzyme, at utmost keep Enzymatic activity.

Use what is formed by NIPA and acrylic or methacrylic acid by copolyreaction in the present invention Carrier material comes immobilizing trypsinase or chymotrypsin, improves the recall rate of enzymolysis efficiency and sample peptide fragment.

According to the present invention, the particle diameter of the temperature sensitive immobilised enzymes microballoon is 0.5-200 μm, for example, can be 0.5 μm, 1 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 120 μm, 150 μm, 180 μm or 200 μm, preferably 1-100 μm, more preferably 1-50 μ m。

Size controlling of the invention by temperature sensitive immobilised enzymes microballoon is at 0.5-200 μm, the temperature sensitive immobilised enzymes under the particle diameter Microballoon, it, which has the advantage that, essentially consists in：It is undersized, such as particle diameter can have stronger Thermo-sensitive less than 400 μm of microballoon, Thermo-sensitive shows rapidly hydrophobic property when hydrophilic microballoon temperature can be caused to reach its critical inversion temperature by force, and then dredges Water microballoon occurs largely to assemble in a short time by hydrophobic interphase interaction, influences to digest；Microballoon, which crosses conference, causes Thermo-sensitive Weaker, hydrophobe fringe time is elongated, influences hydrolysis result.

According to the present invention, the critical inversion temperature (LCST) of the temperature sensitive immobilised enzymes microballoon is 32-45 DEG C, such as can To be 32 DEG C, 33 DEG C, 35 DEG C, 38 DEG C, 40 DEG C, 42 DEG C, 43 DEG C or 45 DEG C, preferably 33-43 DEG C, more preferably 35-40 DEG C.

The present invention controls the critical inversion temperature of temperature sensitive immobilised enzymes microballoon at 32-45 DEG C, this temperature range with The most suitable catalytic temperature of conventional trypsase and chymotrypsin is close.In enzymolysis process, when temperature is the most suitable catalysis temperature of enzyme Degree, while temperature sensitive microballoon shows hydrophobic property, then testing protein can be enriched with to immobilization by hydrophobic interphase interaction Around enzyme microballoon, so as to increase the contact probability of enzyme and albumen, enzymolysis efficiency is improved.

The present invention is using the copolymer microsphere of NIPA and acrylic or methacrylic acid as tryptose The carrier of enzyme immobilization, by adjusting ambient temperature come the hydrophilic and hydrophobic of control vector, the enzymolysis and identification for improving protein are imitated Fruit.In the enzyme digestion reaction stage, by temperature adjustment to more than LCST, make carrier that hydrophobic performance be presented, be effectively enriched with testing protein Sample, improve enzymolysis efficiency.Temperature is reduced to below LCST, carrier is changed into hydrophily, enzymolysis reaction, and discharge Peptide fragment after enzymolysis, so as to improve the recall rate of sample peptide fragment.

According to the present invention, step (1) the thermal denaturation before processing also includes：Protein example to be digested is dissolved in alkali In property solution, specifically, the alkaline solution for example can be ammonium bicarbonate buffers, Tris-HCl buffer solutions or urea liquid Deng not doing particular determination herein；For the concentration of alkaline solution, alkaline solution of the molar concentration in 50-200mmol/L is chosen.

, it is necessary to add go back original reagent and alkylating reagent promotion protein molecule two sulphur of opening during traditional thermal denaturation Key, so that subsequent protein hydrolase is effectively digested to it.And the thermal denaturation process of testing sample is then not required in the present invention Go back original reagent and alkylating reagent are added, the protein sample obtained after denaturation can effectively be digested by temperature sensitive immobilised enzymes.Cause This, the present invention eliminates go back original reagent and alkylating reagent reset procedure, avoids this two classes reagent to subsequent analysis instrument Interference, is also improved the efficiency of denaturation simultaneously because simplifying operating procedure.

According to the present invention, step (1) the thermal denaturation processing procedure is：Under infrared radiation, become in 85-95 DEG C of water-bath Property 1-5min.Compared with simple heating water bath denaturation, aid in being denatured using infrared ray, except system temperature can be lifted, moreover it is possible to Strengthen the vibration of enzyme molecule, improve denaturation efficiency.

For the temperature of thermal denaturation processing, using 85-95 DEG C, such as can be 85 DEG C, 87 DEG C, 88 DEG C, 90 DEG C, 91 DEG C, 92 DEG C, 93 DEG C or 95 DEG C；For the thermal denaturation time, using 1-5min, such as can be 1min, 2min, 3min, 4min or 5min。

Step (1) in the present invention can use following operation：

Need to be dissolved in protein example to be digested in the alkaline solution that molar concentration is 50-200mmol/L first, then Thermal denaturation processing is carried out, under infrared radiation, 1-5min is denatured in 85-95 DEG C of water-bath.

Step (1) can also use the operation not being dissolved in the protein example of enzymolysis in alkaline solution, but directly Protein example to be digested is subjected to thermal denaturation processing under infrared radiation, the temperature and time that it is handled is same as above.

According to the present invention, protein example volume to be digested described in step (1) is 1-20mL, for example, can be 1mL, 2mL, 5mL, 8mL, 10mL, 12mL, 15mL, 18mL, 19mL or 20mL, preferably 1-10mL.Testing protein volume can not mistake It is more, it otherwise can influence degenerative effects.

According to the present invention, the mass ratio of enzyme and testing protein quality sample is 1 in step (2) described enzyme digestion reaction:1-1:40, Such as can be 1:1、1:3、1:5、1:6、1:9、1:10、1:12、1:15、1:18、1:22、1:30、1:35、1:36、1:38 or 1:40, preferably 1:1-1:20.

According to the present invention, the mass concentration of step (2) the testing protein quality sample is 100ng/mL-5mg/mL, such as Can be 100ng/mL, 200ng/mL, 300ng/mL, 1 μ g/mL, 10 μ g/mL, 100 μ g/mL, 1mg/mL, 2mg/mL, 3mg/mL Or 5mg/mL, preferably 0.1 μ g/mL-1mg/mL.

Preferably, the volume of the reaction solution is 1-50mL.The volume of mixed liquor can not be too small, otherwise can make collected The sample volume arrived is less than the sample size of liquid phase-mass spectrometer, and sample can not be detected.Mixed liquor volume can not mistake Greatly, otherwise infrared radiation degree weakens, and can influence hydrolysis result.

In the present invention, the protein solution taking-up part after step (1) is denatured by generally use is come and temperature sensitive immobilization Enzyme microballoon is mixed to form reaction solution.Needed as the protein solution volume taken out according to enzyme in temperature sensitive immobilised enzymes microballoon Calculated with the quality of testing protein quality sample and the mass concentration of testing protein quality sample.

In conventional solution enzymatic isolation method, to reduce the self-enzymatic hydrolysis of enzyme, generally use relatively low enzyme concentration (enzyme-to-substrate quality Than for 1:50) enzyme digestion reaction is carried out.Under this condition in order to reach the purpose fully digested, longer enzymolysis time can be used, Thus it is difficult to solve the problems, such as that the self-enzymatic hydrolysis of enzyme brings background peaks.After immobilization, the stability of enzyme has obtained very big lifting, enzyme Molecule, which meets, occurs the probability reduction of self-digestion.Therefore in enzymolysis process, enzymolysis is improved by suitably increasing the concentration of enzyme Efficiency and the follow-up identification of background peaks interference will not be introduced.Protein substrate concentration selected in enzymolysis process is relatively low, at enzymolysis Selected treatment temperature is higher than the LCST of temperature sensitive immobilised enzymes microballoon during reason, and now immobilised enzymes microballoon can show to dredge Aqueous nature, protein molecular can be enriched in around immobilised enzymes microballoon, therefore can effectively digested by hydrophobic interaction Low concentration protein.

In the present invention, when reaction solution temperature rises to 45-65 DEG C, infrared ray need to be closed.Why by hydrolysis temperature control System within the temperature, in order that make hydrolysis temperature be higher than temperature sensitive immobilised enzymes microballoon critical inversion temperature (LCST), At this temperature, temperature sensitive microballoon shows hydrophobic property, is favorably improved affinity of the immobilised enzymes to protein sample to be measured, and Hydrophobic microballoon can effectively be enriched with testing protein molecule, so as to improve enzymolysis efficiency by hydrophobic interphase interaction.

In the present invention, hydrolysis temperature, if being higher than this temperature, will influence immobilised enzymes to a certain extent not above 65 DEG C Activity, reduce enzymolysis efficiency.

In the enzymolysis process of the present invention, the irradiation of infrared ray can cause the heating of solution, when the temperature increases, can be to enzyme Molecular conformation has an impact, and causes the decline of its enzymolysis activity.But carrier is done using temperature sensing material, can be by absorption system Heat, show hydrophobic state under less than the obvious denaturation temperature of enzyme (45 DEG C), this characteristic provides hydrophobic for enzyme molecule Environment, the molecular conformation of enzyme and the activity of enzyme is set to obtain a certain degree of holding, therefore, infrared radiation and temperature sensitive immobilised enzymes Microballoon has synergistic function between the two, so as to which the efficient enzymolysis of protein is better achieved.

In the enzymolysis process of the present invention, which kind of form of irradiation infrared ray takes according to the volume of reaction solution to determine.Example Such as when system volume is less, the infrared ray used takes intermittent irradiation form, and the alternate form of selection stops 2 to open the 5-20 seconds Second, solution heating rate can be effectively controlled, avoids system temperature from rising sharply.On the other hand, when the volume of system is larger, can use The form of prolonged exposure, ensure infrared assistance enzymolysis effect.When system temperature raises, temperature sensitive carrier can absorb heat and phase occurs Transformation, while hydrophobic microenvironment is provided for enzyme molecule, this plays a certain degree of protective effect to enzyme.Therefore, reaction system When carrying out enzyme digestion reaction, without using external source cooling system.

According to the present invention, the time of step (2) described enzymolysis is 0.5-5min, for example, can be 0.5min, 1min, 2min, 3min, 4min or 5min.

For enzymolysis time used in the present invention less than conventional solution enzymatic isolation method (generally 12-24h), this is due to higher than solid Surely change enzyme microballoon LCST temperature when, hydrophobic microballoon can effective enriched substrates, increase enzymolysis efficiency；Simultaneously because enzyme with Substrate mass ratio is higher, and the increase of enzyme amount directly accelerates enzymolysis speed, therefore can shorten enzymolysis time compared with free enzyme.

In addition, infrared ray can draw the vibration of protein molecular in system, be advantageous to egg in radioreaction system in the present invention The exposure of restriction enzyme site in white molecule, so as to promote enzyme digestion reaction.While infrared radiation, the temperature of reaction solution has Risen, it is microsphere supported to show hydrophobic state when temperature is higher than the LCST of temperature sensitive microballoon, and provide and dredge for protease molecule Water environment, testing protein sample molecule is effectively enriched with by hydrophobic interaction, is effectively increased testing protein quality sample and enzyme Contact probability, therefore can use low concentration testing protein quality sample.Infrared radiation technology and temperature sensitive immobilised enzymes institute shape Into this special synergy, can significantly reduce enzymolysis time within 5min.

According to the present invention, after digesting a period of time, when reaction solution temperature rises to 45-65 DEG C, close infrared ray, need by The temperature of reaction solution is down to 5-25 DEG C, enzymolysis reaction, separates temperature sensitive immobilised enzymes microballoon and enzymolysis product solution, collects Mass Spectrometric Identification is directly carried out after reaction mixture.

, can enzymolysis reaction below the LCST values by cooling the temperature to microballoon after enzyme digestion reaction in the present invention.This When being due to that temperature is less than LCST, the hydrophobic interactions decrease between microballoon performance hydrophilic nmature, with protein sample, directly subtract The touch opportunity of enzyme and albumen is lacked；Temperature declines the activity decrease for also directly resulting in enzyme simultaneously.For these reasons, drop is passed through Low temperature can rapid enzymolysis reaction.Termination procedure does not need additional acidic materials to terminate reaction, effectively avoids Interference of the exogenous agent to sample, while the activity of enzyme is protected, contribute to reusing for immobilised enzymes.In addition, reduce Temperature makes microballoon show another advantage of hydrophilic nmature, and microballoon is in the state of hydrophilic, to the non-specific of albumen and product Property absorption reduce, effectively reaction product can be discharged, improves the peptide fragment recall rate of follow-up mass spectral analysis.Enzyme digestion reaction terminates Afterwards, first pass through centrifugation to separate immobilised enzymes with enzymolysis product, regather the secondary centrifuging that supernatant solution carries out ultrafiltration apparatus, go The removal of impurity and complete protein substrate is not digested, realize the collection of peptide fragment.Because salinity is relatively low in system, therefore collects and obtain Filtrate without desalting processing, can directly carry out the analysis and identification of liquid chromatography-mass spectrography.

Protein digestion method of the present invention can be carried out using following steps：

(1) by protein example to be digested under infrared radiation, 1-5min is denatured in 85-95 DEG C of water-bath；

(2) under infrared radiation, solution is obtained to step (1) using temperature sensitive immobilised enzymes microballoon and carries out enzyme digestion reaction, When the temperature of reaction solution rises to 45-65 DEG C, infrared ray is closed；The matter of enzyme and testing protein quality sample in the enzyme digestion reaction Amount is than being 1:1-1:40；

(3) temperature of reaction solution is down to 5-25 DEG C, enzymolysis reaction, separates temperature sensitive immobilised enzymes microballoon and enzymolysis Reaction mixture, collect enzymolysis product solution.

Present invention also offers foregoing infrared ray-purposes of the temperature sensitive immobilised enzymes microballoon in protein digestion, It is that protein is digested using temperature sensitive immobilised enzymes microballoon under infrared radiation.

The carrier material for the temperature sensitive immobilised enzymes microballoon used in the purposes is by NIPA and acrylic acid Or methacrylic acid is formed by copolyreaction；The immobilised enzymes of temperature sensitive immobilised enzymes microballoon is immobilizing trypsinase or solid Surely chymotrypsin is changed.

When infrared ray and temperature sensitive immobilised enzymes microballoon in the present invention are applied in protein digestion, its specific method is the same as such as Preceding described protein digestion method, will not be described here.

The present invention is heated up to reaction system using infrared ray and promotes the exposure of protease cutting site, can be effectively increased The acting frequency of restriction enzyme site and enzyme, so as to significantly improve enzymolysis efficiency；Meanwhile in system temperature-rise period, temperature sensitive immobilization Simultaneously phase in version occurs for enzyme microballoon absorption system heat, shows hydrophobic property.Hydrophobic microballoon can effectively keep enzymatic activity, and lead to Hydrophobic interaction enrichment testing protein quality sample is crossed, increases the contact probability of enzyme-to-substrate.This infrared ray and immobilised enzymes Coordinated enzymatic hydrolysis acts on, and the efficient enzymolysis of protein can be better achieved, can also effectively avoid because carrier self property and caused by Enzymolysis efficiency it is low, the product release shortcoming that not exclusively causes sample peptide fragment recall rate low etc. after enzymolysis, while preferably solve Enzyme irretrievable problem after use.

Compared with prior art, the present invention at least has the advantages that：

(1) present invention is digested using infrared radiation technology and the synergy of temperature sensitive immobilised enzymes microballoon to protein Processing, realize the efficient enzymolysis of protein；

(2) compared with conventional 37 DEG C of hydrolysis temperature, the present invention can effectively widen hydrolysis temperature scope to 65 DEG C, and realize compared with Protein digestion under high-temperature, on the premise of keeping protein structure stable, improve protein digestion efficiency；

(3) present invention accelerates endonuclease reaction when carrying out protein digestion reaction by using infra-red radiation, while temperature sensitive Carrier absorbs to heat caused by infra-red radiation, and the hydrophobic environment formed can protect enzyme texture image and richness Collect protein, be advantageous to fully contacting for enzyme and testing protein quality sample restriction enzyme site, so as to accelerate the progress digested；

(4) compared with conventional enzymolysis time 12-24h, the time that the present invention completes protein digestion is shorter, can be controlled in 0.5-5min, enzymolysis time is significantly shorten, reduce the consumption of time；

(5) present invention process is simple, safe operation, in the case of without using cooling device, you can using infrared radiation Enzymolysis, reduces the consumption of the energy.

Brief description of the drawings

Fig. 1 is flow chart of the method for the present invention；

Fig. 2 is the total ion current that free enzyme enzymolysis bovine serum albumin(BSA) sample under infrared ray auxiliary obtains reaction mixture Figure, enzymolysis time 1min；

Fig. 3 is total ion that immobilised enzymes enzymolysis bovine serum albumin(BSA) sample under infrared ray auxiliary obtains reaction mixture Flow pattern map, enzymolysis time 1min.

The present invention is described in more detail below.But following examples is only the simple example of the present invention, not generation Table or limitation the scope of the present invention, protection scope of the present invention are defined by claims.

Embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited Scope.

Embodiment 1

A kind of protein digestion method based on immobilised enzymes, its specific operating procedure are as follows：

(1) preparation of temperature sensitive Immobilized chymotrgpsin microballoon

(a) preparation of carrier material

Carrier material presses 9 by NIPA and acrylic acid:1 mass ratio is copolymerized to be formed, using fast Fast membrane emulsification prepares copolymerization microsphere：Acrylic acid, NIPA, initiator, crosslinking agent are added in deionized water, NIPA, initiator, crosslinking agent, the mass ratio of deionized water are 10:0.4:0.4:500, stir complete to solid Fully dissolved, it is configured to aqueous phase；It is 1 in mass ratio by Span-80, hexamethylene:20 mixing, are stirred at room temperature completely molten to Span-80 Solution, is configured to oil phase；It is 1 by volume by oil phase and aqueous phase:5 mixing, 15min is stirred under the slow-speed of revolution and obtains mixed liquor；Will be mixed Close liquid and micropore glass film (aperture is 20 μm) is pressed through under certain nitrogen pressure, obtain water-in-oil emulsion.Water-in-oil emulsion adds In 150mL four-neck flasks, stirred under 140r/min rotating speeds and be passed through nitrogen.Accelerator is added in 1h backward emulsion, at 25 DEG C Lower reaction 6h.Reaction stops stirring after terminating, and microballoon is respectively washed 3 times using acetone and deionized water.Finally cleaning is obtained Microballoon be scattered in deionized water preserve it is stand-by.

(b) preparation of temperature sensitive Immobilized chymotrgpsin microballoon

Temperature sensitive Immobilized chymotrgpsin microballoon is prepared using chemical covalent fixation：Chymotrypsin is dissolved in phosphoric acid buffer In solution (buffer solution ion concentration be 10mmol/L, pH=7.2~7.4), 1mg/mL chymotrypsin solution is configured to.It is another Aspect, microballoon is taken out and is dissolved in cushioning liquid, by accelerator 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt Hydrochlorate is dissolved in 1mL pH=4.0 hydrochloric acid solution, the molal quantity of the carboxylic group contained by the molal quantity and microballoon of accelerator Unanimously, accelerator solution is mixed with microspheres solution, and the mixed liquor is positioned over to the activated carboxylic reaction that 2h is carried out at 25 DEG C. After terminating microballoon is washed with deionized 3~5 times in reaction, removes impurity and is scattered in microballoon in phosphate buffer again.Will Microspheres solution mixes with chymotrypsin solution, and the mass ratio of microballoon and chymotrypsin is 1 in solution:After 12h being reacted at Isosorbide-5-Nitrae DEG C, Immobilised enzymes microballoon is separated with having neither part nor lot in the chymotrypsin of reaction.Finally give the temperature sensitive immobilization gruel albumen that particle diameter is 25 μm Enzyme microballoon, the enzyme amount fixed are 200mg/g than the quality of microballoon, and its LCST value is 38 DEG C.Resulting immobilization gruel albumen Enzyme microballoon is scattered in phosphate buffer solution, is saved backup at 4 DEG C.

(2) cromoci sample to be digested is dissolved in 95 DEG C of 100mM NH₄HCO₃In solution, liquor capacity is 10mL, carries out thermal denaturation processing, processing time 5min under 50W infrared rays to cromoci sample, and processing will become after terminating The cromoci sample solution of property is cooled to room temperature.

(3) under the auxiliary of the infrared ray, the degeneration of cells pigment C using temperature sensitive Immobilized chymotrgpsin microballoon to gained Sample carries out enzymolysis processing, and the concentration of cromoci sample be 0.5mg/mL, and the mass ratio of enzyme and cromoci is 1:10, from Liquor capacity is 15mL in heart pipe.The alternate form of infrared line options is stopped 2 seconds for 15 seconds to open, enzymolysis time 5min.

(4) cool the temperature to room temperature and centrifuge 10min under 4000r/min simultaneously, enzyme digestion reaction is terminated, received respectively Collect immobilised enzymes microballoon and supernatant solution.Supernatant solution is added in 10kDa ultra-filtration centrifuge tubes, centrifuged under 5000r/min 20min, filtrate is collected, the instrument for then carrying out following liquid-phase chromatography-mass spectroscopy series connection is analyzed.First by liquid chromatogram pair Sample separated (chromatographic systems of Agilent 1100, Thermo scientific Snycronis-C18 posts (4.6 × 250mm, 5 μm)；Mobile phase：A, H₂O (0.1%TFA)；B, ACN (0.1%TFA)；Condition of gradient elution：0-70min, 5%- 70%B；Flow velocity：The μ L of 0.75mL/min sample sizes 70.Then Tandem Mass Spectrometry Analysis, Mass Spectrometry Conditions are carried out：LCQ DECAXP ions Trap mass spectrum, ion gun (ESI) spray voltage are 4.5kV；Capillary temperature is 300 DEG C；60 units of sheath gas (N2)；Cation side Formula detects, and is detected using full scan first mass spectrometric, first mass spectrometric scanning range m/z 300-2000, the scanning of accurate mass number The scanning of (Zoom scan) and second order mses (MS/MS) is data dependence type scanning (Data Dependent Scan)；Dynamically Exclude number 1；Dynamic excludes time 0.5min；Second order mses collision energy is 35%.The initial data that mass spectrum is obtained uses Turbosequenst softwares scan for.

Resulting peptide segment data is compared, as a result as shown in table 1.Under 37 DEG C of insulating box environment, freely After enzyme lysed cells pigment C 10h, the matching peptide hop count detected is 6.When under infrared auxiliary, free enzyme digests 5min When, the matching peptide hop count that can be detected is 5, under identical infrared ray condition and enzymolysis time, immobilised enzymes lysed cells The peptide fragment bar number detected in pigment C reaction mixture is 10, illustrates to utilize the infrared ray in the present invention and immobilised enzymes association With enzymolysis, enzymolysis efficiency and peptide fragment recall rate can be effectively improved.

Table 1

Sample	Heating condition	Time	Match peptide hop count
				Free enzyme	37 DEG C of water-baths	10h	6
Free enzyme	50W is infrared	5min	5
				Immobilised enzymes	50W is infrared	5min	10

Embodiment 2

A kind of protein digestion method based on immobilised enzymes, its specific operating procedure are as follows：

(1) preparation of temperature sensitive immobilizing trypsinase microballoon

(a) preparation of carrier material

Carrier material presses 6 by NIPA and methacrylic acid:1 mass ratio is copolymerized to be formed, and is adopted Copolymerization microsphere is prepared with microflow control technique：NIPA, mixed using n-hexane-acetone (volume ratio 50/50) molten Agent recrystallizes 3 times, comonomer acrylic acid, removes polymerization inhibitor using 5% NaOH solution, initiator is ammonium persulfate, crosslinking Agent is N, and N '-methylene-bisacrylamide, accelerator is tetramethylethylenediamine.By methacrylic acid, NIPA, Initiator, crosslinking agent are added in deionized water, and NIPA, initiator, crosslinking agent, the mass ratio of deionized water are 10: 0.6: 1.5: 100, stir to solid and be completely dissolved, be configured to aqueous phase；Arlacel-80, decyl alcohol is mixed in mass ratio for 1: 10 Close, be stirred at room temperature to Span-80 and be completely dissolved, be configured to oil phase；Aqueous phase solution is with certain speed (100 μ L/h) from micro-fluidic dress Put in the injection-tube (internal diameter is 400 μm) in chip and pass through；Injection-tube is imported below oil phase liquid level, aqueous phase solution is passed through completely Water-in-oil emulsion is can obtain after to oil-phase solution.Water-in-oil emulsion is added in 150mL four-neck flasks, and stirring at low speed is simultaneously passed through nitrogen Gas.Accelerator is added in 1h backward emulsion, reacts 6h at 25 DEG C.Reaction terminate after stop stirring, using acetone and go from Sub- water is respectively washed microballoon 3 times.To finally clean obtained microballoon be scattered in deionized water preserve it is stand-by.

(b) preparation of temperature sensitive immobilizing trypsinase microballoon

Temperature sensitive immobilizing trypsinase microballoon is prepared using the method for physical absorption and chemical covalent secure bond：

1.1 adsorption reaction

Trypsase is dissolved in phosphate buffer solution (buffer solution ion concentration is 10mmol/L, pH=7.2~ 7.4), it is configured to 1mg/mL trypsin solution.Microballoon is taken out simultaneously to add in trypsin solution, microballoon and trypsase Mass ratio is 1:1.5, mixed liquor carries out 2h adsorption reaction at 4 DEG C.

1.2 is covalently fixed

Accelerator is added in 1mL pH=4.0 hydrochloric acid solution, add contain microballoon and tryptose until completely dissolved In the solution of enzyme, the molal quantity of accelerator is consistent with the molal quantity of the carboxylic group contained by microballoon, and the mixed liquor is positioned over into 25 2h covalent reaction is carried out at DEG C, then continues to react 10h at 4 DEG C.Reaction terminates, and by immobilised enzymes microballoon and has neither part nor lot in anti- Activator, the trypsase answered separate.The temperature sensitive immobilizing trypsinase microballoon that particle diameter is 200 μm is finally given, is fixed Enzyme amount is 410mg/g than the quality of microballoon, and its LCST value is 45 DEG C.Resulting immobilizing trypsinase microballoon is scattered in phosphoric acid In cushioning liquid, saved backup at 4 DEG C.

(2) bovine serum albumin(BSA) (BSA) sample to be digested is dissolved in 85 DEG C of 200mM NH₄HCO₃In solution, solution Volume is 15mL, carries out thermal denaturation processing, processing time 1min to BSA samples under 400W infrared rays, and processing will after terminating The BSA samples of denaturation are cooled to room temperature.

(3) while under the auxiliary of the infrared ray, using denaturation BSA sample of the temperature sensitive immobilization proteinase pancreas microballoon to gained Product carry out enzymolysis processing, and the concentration of BSA samples be 5mg/mL, and enzyme and BSA mass ratio are 1:40, liquor capacity is in centrifuge tube 50mL.The form of infrared line options continuous heating, enzymolysis time 0.5min.

(4) cool the temperature to room temperature and centrifuge 5min under 5000r/min simultaneously, enzyme digestion reaction is terminated, received respectively Collect immobilised enzymes microballoon and supernatant solution.Supernatant solution is added in 50kDa ultra-filtration centrifuge tubes, centrifuged under 12000r/min 5min, filtrate is collected, the instrument for then carrying out following liquid-phase chromatography-mass spectroscopy series connection is analyzed.Liquid chromatography-mass spectrography series connection instrument The relevant parameter of device analysis is the same as embodiment 1.

Fig. 2 is digested the parent ion stream that bovine serum albumin(BSA) sample obtains reaction mixture by free enzyme under infrared ray auxiliary Figure, Fig. 3 are digested the parent ion flow graph that bovine serum albumin(BSA) sample obtains product by immobilised enzymes under infrared ray auxiliary.In figure The quasi-molecular ions of molecule is corresponded in the presence of some peptide hydrolysis, by the secondary fragment information input data storehouse of these parent ions, passes through letter The search and comparison of breath, find from free enzyme digest after sample in detect that matching peptide hop count is 6, and from immobilised enzymes enzyme It is 15 that matching peptide hop count is detected in sample after solution, and this explanation is by using infrared ray and immobilization proposed by the invention The synergy of enzyme carries out the enzymolysis of protein, can greatly strengthen the efficiency of enzymolysis.

Embodiment 3

A kind of protein digestion method based on immobilised enzymes, its specific operating procedure are as follows：

(1) preparation of temperature sensitive immobilizing trypsinase microballoon

(a) preparation of carrier material

Carrier material presses 1 by NIPA and acrylic acid:1 mass ratio is copolymerized to be formed, using nothing Soap emulsion polymerization prepares copolymerization microsphere：By monomer NIPA, acrylic acid and crosslinking agent are dissolved in 200mL and gone It is put into after ionized water in 250mL four-neck flask, NIPA, crosslinking agent, the mass ratio of deionized water are 100: 0.3:20000 are passed through nitrogen and carry out mechanical agitation, and initiator is added after stable 15min, and temperature is increased into 80 DEG C, continue anti- Answer 4h.Reaction cools the temperature to room temperature after terminating, and sample is fitted into molecular cut off 20000-50000 bag filter in room temperature Lower pure water dialysis 48-72h.Finally, sample is freezed, be sealed at room temperature.

(b) preparation of temperature sensitive immobilizing trypsinase microballoon

Temperature sensitive immobilizing trypsinase microballoon is prepared using chemical covalent method：Trypsase is dissolved in phosphate buffer solution In (buffer solution ion concentration is 10mmol/L, pH=7.4), be configured to 1mg/mL trypsin solution.On the other hand, will be micro- Ball takes out and is dissolved in cushioning liquid, by accelerator 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine hydrochloride salt In 1mL pH=4.0 hydrochloric acid solution, the molal quantity of accelerator is consistent with the molal quantity of the carboxylic group contained by microballoon, will Accelerator solution is mixed with microspheres solution, and the mixed liquor is positioned over to the activated carboxylic reaction that 2h is carried out at 25 DEG C.Reaction knot Microballoon is washed with deionized after beam 3~5 times, removes impurity and be scattered in microballoon in phosphate buffer again.Microballoon is molten Liquid mixes with trypsin solution, and the mass ratio of microballoon and trypsase is 2 in solution:After reacting 12h at Isosorbide-5-Nitrae DEG C, by fixation Change enzyme microballoon to separate with having neither part nor lot in the trypsase of reaction.It is micro- to finally give the temperature sensitive immobilizing trypsinase that particle diameter is 0.5 μm Ball, the enzyme amount fixed are 300mg/g than the quality of microballoon, and its LCST value is 36 DEG C.Resulting immobilizing trypsinase is micro- Ball is scattered in phosphate buffer solution, is saved backup at 4 DEG C.

(2) myoglobins sample to be digested is dissolved in 90 DEG C of 100mM ammonium bicarbonate buffer solutions, liquor capacity For 4mL, thermal denaturation processing, processing time 3min are carried out to protein solution under 275W infrared rays, processing will become after terminating The protein example of property is cooled to room temperature.

(3) under the auxiliary of the infrared ray, using denaturation myoglobins of the temperature sensitive microsphere immobilized trypsase to gained Sample carries out enzymolysis processing, and the concentration of myoglobins sample be 100ng/mL, and the mass ratio of enzyme and myoglobins is 1:1, centrifugation Liquor capacity is 1mL in pipe.The alternate form of infrared line options is stopped 2 seconds for 5 seconds to open, enzymolysis time 3min.

(4) 20min is centrifuged under 8000rpm while cooling the temperature to room temperature, enzyme digestion reaction is terminated, is received respectively Collect immobilised enzymes microballoon and supernatant solution.Supernatant solution is added in 10kDa ultra-filtration centrifuge tubes, centrifuged under 9000r/min 15min, filtrate is collected, the instrument for then carrying out following liquid-phase chromatography-mass spectroscopy series connection is analyzed.Liquid chromatography-mass spectrography is connected For the relevant parameter of Instrumental Analysis with embodiment 1, the peptide fragment bar number detected in its reaction mixture is 9.

Embodiment 4

Compared with Example 3, except in carrier material the mass ratio of NIPA and acrylic acid be 20:Outside 1, It is other same as Example 3.Finally give the temperature sensitive immobilizing trypsinase microballoon that particle diameter is 0.5 μm, the enzyme amount ratio fixed The quality of microballoon is 100mg/g, and its LCST value is 34 DEG C.

(2) follow-up enzymolysis step is consistent with embodiment 3, and the peptide fragment bar number detected in enzymolysis product solution is 5.

Embodiment 5

Compared with Example 3, except in carrier material the mass ratio of NIPA and acrylic acid be 3:Outside 1, It is other same as Example 3.Finally give the temperature sensitive immobilizing trypsinase microballoon that particle diameter is 0.5 μm, the enzyme amount ratio fixed The quality of microballoon is 240mg/g, and its LCST value is 35 DEG C.

(2) follow-up enzymolysis step is consistent with embodiment 3, and the peptide fragment bar number detected in enzymolysis product solution is 12.

Embodiment 6

Compared with Example 3, it is other same as Example 3 in addition to the specific preparation method difference of carrier material.It has The preparation method of body is precipitation polymerization method：By acrylic acid, NIPA, initiator, crosslinking agent solvent are dissolved in In the 80mL aqueous solution, the concentration of NIPA is 8g/L；NIPA, initiator, crosslinking in solution The mass ratio of agent is 10:0.4:1；Lead to nitrogen 30min into solution, then heat above-mentioned solution under oil bath or water bath condition To 70 DEG C, 12h is reacted；React and reaction solution temperature is down to room temperature after terminating, sample is loaded into molecular cut off 20000- In 50000 bag filter at room temperature pure water dialysis 72h.Finally, sample is freezed, be sealed at room temperature.Prepare Thermo-sensitive The method of immobilised enzymes is consistent with embodiment 3, finally gives the temperature sensitive immobilizing trypsinase microballoon that particle diameter is 0.8 μm, consolidate Fixed enzyme amount is 300mg/g than the quality of microballoon, and its LCST value is 37 DEG C.

(2) follow-up enzymolysis step is consistent with embodiment 3, and the peptide fragment bar number detected in enzymolysis product solution is 10.

Embodiment 7

(1) compared with Example 3, it is other same as Example 3 in addition to the specific preparation method difference of carrier material. Its specific preparation method is dispersion copolymerization method：5g stabilizers are added in 50mL water and stirred, by acrylic acid, N- isopropyls Acrylamide, crosslinking agent are added thereto and are passed through nitrogen.NIPA, crosslinking agent, the mass ratio of deionized water are 1:0.5:80.System temperature is risen to 60 DEG C after 30min, 0.5g initiators is added after stable 15min, reacts 12h.Reaction terminates After cool the temperature to room temperature, by sample be fitted into molecular cut off 20000-50000 bag filter at room temperature pure water dialysis 72h.Finally, sample is freezed, be sealed at room temperature.The method for preparing Thermo-sensitive immobilised enzymes is consistent with embodiment 3, finally The temperature sensitive immobilizing trypsinase microballoon that particle diameter is 0.5 μm is obtained, the enzyme amount fixed is 290mg/g than the quality of microballoon, its LCST values are 35 DEG C.

(2) follow-up enzymolysis step is consistent with embodiment 3, and the peptide fragment bar number detected in enzymolysis product solution is 9.

Embodiment 8

Compared with Example 3, in addition to the preparation method difference of temperature sensitive immobilizing trypsinase microballoon, other and embodiment 3 is identical.Temperature sensitive immobilizing trypsinase microballoon is prepared using the method for physical absorption and chemical covalent secure bond.

1.1 adsorption reaction

Trypsase is dissolved in phosphate buffer solution (buffer solution ion concentration is 10mmol/L, pH=7.2~ 7.4), it is configured to 1mg/mL trypsin solution.Microballoon is taken out simultaneously to add in trypsin solution, microballoon and trypsase Mass ratio is 1:1, mixed liquor carries out 2h adsorption reaction at 4 DEG C.

1.2 is covalently fixed

Accelerator is added in 1mL pH=4.0 hydrochloric acid solution, add contain microballoon and tryptose until completely dissolved In the solution of enzyme, the molal quantity of accelerator is consistent with the molal quantity of the carboxylic group contained by microballoon, and the mixed liquor is positioned over into 25 2h covalent reaction is carried out at DEG C, then continues to react 10h at 4 DEG C.Reaction terminates, and by immobilised enzymes microballoon and has neither part nor lot in anti- Activator, the trypsase answered separate.The temperature sensitive immobilizing trypsinase microballoon that particle diameter is 0.5 μm is finally given, is fixed Enzyme amount is 320mg/g than the quality of microballoon, and its LCST value is 37 DEG C.Resulting immobilizing trypsinase microballoon is scattered in phosphoric acid In cushioning liquid, saved backup at 4 DEG C.

(2) follow-up enzymolysis step is consistent with embodiment 3, and the peptide fragment bar number detected in enzymolysis product solution is 15.

Embodiment 9

(1) compared with Example 3, in addition to the preparation method difference of temperature sensitive immobilizing trypsinase microballoon, it is other with it is real It is identical to apply example 3.Temperature sensitive immobilizing trypsinase microballoon is prepared using physisorphtion：It is molten that trypsase is dissolved in phosphoric acid buffer In liquid (buffer solution ion concentration is 10mmol/L, pH=7.2), 1mg/mL trypsin solution is configured to.Take out microballoon simultaneously Add in trypsin solution, the mass ratio of microballoon and trypsase is 1:2, mixed liquor carries out 12h adsorption reaction at 4 DEG C. After reaction terminates, immobilizing trypsinase microballoon is separated with having neither part nor lot in the trypsase of adsorption reaction, finally giving particle diameter is 0.5 μm of temperature sensitive immobilizing trypsinase microballoon, the enzyme amount fixed are 110mg/g than the quality of microballoon, and its LCST value is 35 ℃.Immobilizing trypsinase microballoon is scattered in phosphate buffer solution, is saved backup at 4 DEG C.

(2) follow-up enzymolysis step is consistent with embodiment 3, and the peptide fragment bar number detected in enzymolysis product solution is 4.

Comparative example 1

Compared with Example 3, it is other with the phase of embodiment 3 in addition to immobilised enzymes is replaced with into immobilized papain Together.The temperature sensitive immobilized papain microballoon that particle diameter is 0.5 μm is finally given, the enzyme amount fixed is than the quality of microballoon 240mg/g, its LCST value are 35 DEG C.The peptide fragment bar number detected in enzymolysis product solution is 3.

Comparative example 2

Compared with Example 3, it is other same as Example 3 in addition to immobilised enzymes is replaced with into immobilized lipase.Most The temperature sensitive immobilized papain microballoon that particle diameter is 0.5 μm is obtained eventually, and the enzyme amount fixed is 240mg/ than the quality of microballoon G, its LCST value are 35 DEG C.The peptide fragment bar number detected in enzymolysis product solution is 5.

Comparative example 3

Compared with Example 3, it is other with implementing in addition to the temperature sensitive microballoon of carrier material is replaced with into graphene oxide composite material Example 3 is identical.Follow-up enzymolysis step is consistent with embodiment 3, and the peptide fragment bar number detected in enzymolysis product solution is 6.

Comparative example 4

Compared with Example 3, it is other with the phase of embodiment 3 in addition to the temperature sensitive microballoon of carrier material is replaced with into silica Together.Follow-up enzymolysis step is consistent with embodiment 3, and the peptide fragment bar number detected in enzymolysis product solution is 3.

Comparative example 5

Compared with Example 3, it is other with the phase of embodiment 3 except step (2) is using in addition to heating and substituting infrared radiation Together.Follow-up enzymolysis step is consistent with embodiment 3, and the peptide fragment bar number detected in enzymolysis product solution is 1.

In summary, infrared ray provided by the invention-immobilised enzymes coordinated enzymatic hydrolysis method of protein, is greatly improved enzyme Efficiency and the recall rate of sample peptide fragment are solved, operating procedure simply, conveniently, can be widely used for grinding for the association areas such as proteomics Study carefully.

Applicant states that the present invention illustrates the detailed construction feature of the present invention by above-described embodiment, but the present invention is simultaneously Above-mentioned detailed construction feature is not limited to, that is, does not mean that the present invention has to rely on above-mentioned detailed construction feature and could implemented.Institute Belong to those skilled in the art it will be clearly understood that any improvement in the present invention, to the equivalence replacement of part selected by the present invention And the increase of accessory, selection of concrete mode etc., all fall within protection scope of the present invention and it is open within the scope of.

The preferred embodiment of the present invention described in detail above, still, the present invention are not limited in above-mentioned embodiment Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, this A little simple variants belong to protection scope of the present invention.

It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can The combination of energy no longer separately illustrates.

In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally The thought of invention, it should equally be considered as content disclosed in this invention.

Claims (10)

1. A kind of 1. method of protein digestion, it is characterised in that it includes：It is micro- using temperature sensitive immobilised enzymes under infrared radiation Ball digests to protein.
2. 2. the method as described in claim 1, it is characterised in that it comprises the following steps：

   (1) protein example to be digested is subjected to thermal denaturation processing；

   (2) under infrared radiation, the solution obtained using temperature sensitive immobilised enzymes microballoon to step (1) carries out enzyme digestion reaction, when When the temperature of reaction solution rises to 45-65 DEG C, infrared ray is closed；

   (3) temperature of reaction solution is down to 5-25 DEG C, enzymolysis reaction, separates temperature sensitive immobilised enzymes microballoon and enzymolysis product Solution, collect enzymolysis product solution.
3. 3. method as claimed in claim 1 or 2, it is characterised in that the power of the infrared ray is 50-400W, and wavelength is 2.5-20μm；

   Preferably, the carrier material of the temperature sensitive immobilised enzymes microballoon is by NIPA and acrylic acid or methyl-prop Olefin(e) acid is formed by copolyreaction, is preferably formed by NIPA with acrylic acid by copolyreaction；

   Preferably, the immobilised enzymes of the temperature sensitive immobilised enzymes microballoon is immobilizing trypsinase or Immobilized chymotrgpsin, excellent Elect immobilizing trypsinase as.
4. 4. the method as described in one of claim 1-3, it is characterised in that the particle diameter of the temperature sensitive immobilised enzymes microballoon is 0.5- 200 μm, preferably 1-100 μm, more preferably 1-50 μm；

   Preferably, the critical inversion temperature of the temperature sensitive immobilised enzymes microballoon be 32-45 DEG C, preferably 33-43 DEG C, more preferably For 35-40 DEG C.
5. 5. the method as described in one of claim 2-4, it is characterised in that step (1) the thermal denaturation before processing also includes：Will Protein example to be digested is dissolved in alkaline solution；

   Preferably, the alkaline solution is any one in ammonium bicarbonate buffers, Tris-HCl buffer solutions or urea liquid Kind, preferably urea liquid；

   Preferably, the molar concentration of the alkaline solution is 50-200mmol/L.
6. 6. the method as described in one of claim 2-5, it is characterised in that step (1) the thermal denaturation processing procedure is：Red Under UV radiation, 1-5min is denatured in 85-95 DEG C of water-bath；

   Preferably, protein example volume to be digested described in step (1) is 1-20mL, preferably 1-10mL.
7. 7. the method as described in one of claim 2-6, it is characterised in that enzyme and egg to be measured in step (2) described enzyme digestion reaction The mass ratio of white matter sample is 1:1-1:40, preferably 1:1-1:20；

   Preferably, the mass concentration of the testing protein quality sample is 100ng/mL-5mg/mL, preferably 0.1 μ g/mL-1mg/ mL；

   Preferably, the volume of the reaction solution is 1-50mL；

   Preferably, the time of the enzyme digestion reaction is 0.5-5min.
8. 8. the method as described in one of claim 2-7, it is characterised in that the temperature of step (3) described enzymolysis reaction is 5-25 DEG C, preferably 5-20 DEG C, more preferably 5-10 DEG C.
9. 9. the method as described in one of claim 1-8, it is characterised in that the described method comprises the following steps：

   (1) by protein example to be digested under infrared radiation, 1-5min is denatured in 85-95 DEG C of water-bath；

   (2) under infrared radiation, solution is obtained to step (1) using temperature sensitive immobilised enzymes microballoon and carries out enzyme digestion reaction, when anti- When answering the temperature of solution to rise to 45-65 DEG C, infrared ray is closed；The mass ratio of enzyme and testing protein quality sample in the enzyme digestion reaction For 1:1-1:40；

   (3) temperature of reaction solution is down to 5-25 DEG C, enzymolysis reaction, separates temperature sensitive immobilised enzymes microballoon and enzymolysis product Solution, collect enzymolysis product solution.
10. 10. infrared ray-purposes of the temperature sensitive immobilised enzymes microballoon in protein digestion, it is characterised in that under infrared radiation Protein is digested using temperature sensitive immobilised enzymes microballoon；

    Preferably, the carrier material of the temperature sensitive immobilised enzymes microballoon is by NIPA and acrylic acid or methyl-prop Olefin(e) acid is formed by copolyreaction, is preferably formed by NIPA with acrylic acid by copolyreaction；

    Preferably, the immobilised enzymes of the temperature sensitive immobilised enzymes microballoon is immobilizing trypsinase or Immobilized chymotrgpsin, excellent Elect immobilizing trypsinase as.