CN111521666B - Rapid hydrolysis analysis method for protein under high-temperature and high-pressure state - Google Patents
Rapid hydrolysis analysis method for protein under high-temperature and high-pressure state Download PDFInfo
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Abstract
The invention discloses a rapid hydrolysis analysis method for protein under a high-temperature and high-pressure state. Wherein, the method comprises the following steps: (1) mixing a protein sample with acid to prepare an acidic solution, wherein the protein sample contains aspartic acid; (2) heating the acidic solution to a high temperature and high pressure state in a closed environment so as to hydrolyze the protein sample; (3) and (3) performing mass spectrometry on the hydrolysate obtained in the step (2) by using a high-resolution mass spectrometer. The method has the advantages of simple steps, rapid treatment, less introduced impurities and low cost, and can obtain original protein sequence information by identifying the specific hydrolysis fragments of the protein.
Description
Technical Field
The invention belongs to the field of proteomics research, and particularly relates to a method for detecting characteristic polypeptide fragments of proteins from bottom to top, and more particularly relates to a method for rapidly hydrolyzing and analyzing proteins under a high-temperature and high-pressure state.
Background
The method of using protease to specifically dissociate protein and detecting newly generated free characteristic peptide fragment is one of the most common protein analysis methods. The sites of action of different types of proteases are different, the most common sites of action of trypsin are the C-terminal ends of lysine and arginine. The traditional enzymolysis usually needs a proper environment temperature (about 37 ℃) and a long reaction time (more than 10 hours), and because the introduced reaction system is complex and has more impurities, the separation and purification are carried out by using liquid chromatography and other modes, or the mass spectrum sampling is carried out by using a matrix-assisted laser dissociation technology. In recent years, the novel enzyme cleavage technologies such as immobilized protease technology and microwave catalytic protease technology improve the protein dissociation efficiency to a certain extent, but still have the disadvantages of poor device repeatability, complex reaction system and introduction of more impurities such as macromolecular organic matters (such as protease autolytic fragments) and small molecular inorganic salts.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a rapid hydrolysis analysis method for protein under high temperature and high pressure, which has simple steps, rapid treatment and less introduced impurities, and is a novel bottom-up protein analysis method capable of obtaining original protein sequence information by identifying specific hydrolysis fragments of protein.
According to a first aspect of the present invention, the present invention provides a rapid hydrolysis analysis method for protein under high temperature and high pressure. According to an embodiment of the invention, the method comprises:
(1) mixing a protein sample with acid to prepare an acidic solution, wherein the protein sample contains aspartic acid;
(2) heating the acidic solution to a high temperature and high pressure state in a closed environment so as to hydrolyze the protein sample;
(3) and (3) performing mass spectrometry on the hydrolysate obtained in the step (2) by using a high-resolution mass spectrometer.
The inventor finds that the protein is heated in an acidic environment, and the C-terminal site and the N-terminal site of aspartic acid on a peptide chain of the protein can be broken without introducing protease, so that macromolecular organic matters such as protease autolysate and the like can be avoided, and non-volatile impurities which are not beneficial to mass spectrometric detection, such as inorganic salts and the like existing in the preparation process of the protease can be avoided; compared with the conventional enzymatic hydrolysis immobilized enzyme technology and the like, the method does not need to carry out pretreatment on a hydrolysis reaction device, and the hydrolysis device is simple to build and has good repeatability; in addition, compared with the conventional enzymolysis temperature of 37 ℃ and the conventional enzymolysis time of 12-24h, the method has the advantages that the protein is hydrolyzed under the high-temperature and high-pressure state, the enzymolysis efficiency is greatly improved, and the time consumption is remarkably reduced. In summary, the rapid hydrolysis analysis method for protein under high temperature and high pressure condition of the embodiments of the present invention not only has simple steps, rapid processing, less introduced impurities and low cost, but also can obtain original protein sequence information by identifying specific hydrolysis fragments of protein, and specifically, the bottom-up protein analysis method can be used as a powerful tool for analyzing protein characteristic peptide fragments and fingerprint, thereby specifically identifying target protein.
In addition, the rapid hydrolysis analysis method for protein under high temperature and high pressure according to the above embodiment of the present invention may further have the following additional technical features:
in some embodiments of the present invention, in step (1), the pH value of the acidic solution is 1 to 5, preferably 2 to 3.
In some embodiments of the present invention, the protein sample is recombinant human ubiquitin protein, bovine serum albumin, recombinant tuberculosis antigen Esat-6 protein, recombinant tuberculosis antigen Cfp-10 protein, ribonuclease or horseradish catalase, etc.
In some embodiments of the present invention, in step (1), the protein sample is mixed with a reducing agent in advance before the acidic solution is prepared, so as to cleave disulfide bonds in the protein sample.
In some embodiments of the present invention, in the step (2), the heating time is not more than 20min, preferably 1-4 min.
In some embodiments of the present invention, in the step (2), the temperature of the acidic solution in the high-temperature and high-pressure state is 105 to 200 ℃, and the pressure is 1.2 to 16 atm.
In some embodiments of the present invention, in the step (2), the temperature of the acidic solution in the high-temperature and high-pressure state is 150 to 200 ℃ and the pressure is 5 to 16 atm.
In some embodiments of the present invention, in the step (3), the hydrolysate obtained in the step (2) is mixed with a polar solvent in advance and then analyzed by a high resolution mass spectrometer.
In some embodiments of the invention, in the step (3), the mass concentration of the protein in the hydrolysate is 1-200 ppm.
In some embodiments of the invention, in the step (3), the sample injection manner of the mass spectrometry is a nano-flow electrospray ion source.
In some embodiments of the present invention, in step (2), the acidic solution is heated in a micro-closed reaction vessel, which is a micro-capillary reactor or a micro-high-pressure reactor.
In some embodiments of the present invention, in the microcapillary reactor, the capillary is made of a flexible capillary or a rigid capillary.
In some embodiments of the present invention, the capillary is an elongated capillary, one end of the capillary absorbs the acidic solution by connecting to a syringe or a syringe pump, and the two ends of the capillary are sealed by pressurizing.
In some embodiments of the invention, the capillary tube has an inner diameter of no greater than 4mm and a length of no greater than 20 cm; preferably, the capillary tube has an inner diameter of no more than 2mm and a length of no more than 10 cm.
In some embodiments of the present invention, the capillary is a high molecular polymer capillary, and the capillary is sealed by radial pressure clamping; or the capillary tube is a stainless steel capillary tube, and the capillary tube is sealed in an axial pressurizing and plugging mode.
In some embodiments of the invention, the heating is by contact heating or non-contact heating;
in some embodiments of the present invention, the heating is performed by a non-contact heating method, and a heat conducting material is filled between the outer wall of the capillary tube and the heat generating unit.
According to a second aspect of the present invention, the present invention provides a micro-capillary reactor for use in the above-mentioned rapid hydrolysis analysis method of protein under high temperature and high pressure conditions. According to an embodiment of the present invention, the microcapillary reactor comprises:
a heated capillary, the heated capillary further comprising:
a capillary tube;
the valve comprises a first valve and a second valve, the first valve and the second valve are respectively arranged at two ends of the capillary tube, and the first valve and the second valve control the closed state of the capillary tube;
the metal pipe is sleeved outside the capillary and positioned between the first valve and the second valve, and heat conduction materials are filled between the inner surface of the metal pipe and the outer surface of the capillary;
the heating wire is wound on the outer surface of the metal pipe, and an insulating layer is coated on the surface of the heating wire;
the thermistor is arranged in the middle of the metal tube and is in contact with the metal tube through a small hole in the surface of the insulating layer;
the heating controller comprises a controller and a power amplifier, and the controller is connected with the heating capillary tube through the power amplifier.
According to the micro capillary reactor of the embodiment of the invention, when the acid solution is heated, the capillary tube can be used for containing the acid solution, the capillary tube containing the acid solution is sealed by the first valve and the second valve, the acid solution in the capillary tube is indirectly heated by the heating wire through the metal tube, the heat conduction material is filled between the inner surface of the metal tube and the outer surface of the capillary tube to improve the heat transfer efficiency, the thermistor is used for feeding back the real-time heating temperature, and the power amplifier receives a signal of the controller to control the channel of the circuit so as to start and stop the heating of the capillary tube. Therefore, the acidic solution can be quickly brought into a high-temperature and high-pressure state, so that the quick hydrolysis of the protein is realized.
In some embodiments of the present invention, the capillary is a high molecular polymer capillary, the valve includes a screw and a clamping tube, the clamping tube has a thread matching with the screw, the clamping tube is sleeved at two ends of the capillary, and the screw is connected to the clamping tube through the thread.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a flowchart of a rapid hydrolysis analysis method of protein under high temperature and high pressure conditions according to an embodiment of the present invention.
FIG. 2 is a mechanism diagram of rapid hydrolysis of protein under high temperature and high pressure conditions according to an embodiment of the present invention.
Fig. 3 is a schematic structural view of a microcapillary reactor according to one embodiment of the present invention.
FIG. 4 is an amino acid fingerprint of recombinant human ubiquitin protein according to embodiment 1 of the invention.
FIG. 5 is a mass spectrum of the hydrolysis result of the recombinant human ubiquitin protein corresponding to FIG. 4 according to example 1 of the present invention.
FIG. 6 is an amino acid fingerprint of bovine serum albumin according to example 2 of the present invention.
FIG. 7 is a mass spectrum of the result of hydrolysis of bovine serum albumin according to example 2 of the present invention corresponding to FIG. 6.
FIG. 8 is an amino acid fingerprint of recombinant tuberculosis antigen Esat-6 protein according to example 3 of the present invention.
FIG. 9 is a mass spectrum of the hydrolysis result of the recombinant tuberculosis antigen Esat-6 protein corresponding to FIG. 8 according to example 3 of the present invention.
FIG. 10 is an amino acid fingerprint of recombinant tuberculosis antigen Cfp-10 protein according to example 4 of the present invention.
FIG. 11 is a mass spectrum of the hydrolysis result of the recombinant tuberculosis antigen Cfp-10 protein corresponding to FIG. 8 according to example 4 of the present invention.
FIG. 12 is a mass spectrum of the hydrolysis result of the recombinant human ubiquitin protein corresponding to FIG. 4 in example 5 of the present invention.
FIG. 13 is a mass spectrum of the hydrolysis result of the recombinant human ubiquitin protein corresponding to FIG. 4 in example 6 of the present invention.
FIG. 14 is a mass spectrum of the hydrolysis result of the recombinant human ubiquitin protein of comparative example 1 corresponding to FIG. 4 according to the present invention.
FIG. 15 is a mass spectrum of the hydrolysis result of the recombinant human ubiquitin protein of comparative example 2 corresponding to FIG. 4 according to the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
According to a first aspect of the present invention, the present invention provides a rapid hydrolysis analysis method for protein under high temperature and high pressure. According to an embodiment of the invention, with reference to fig. 1, the method comprises: (1) mixing a protein sample with acid to prepare an acidic solution, wherein the protein sample contains aspartic acid; (2) heating the acidic solution to a high-temperature high-pressure state in a closed environment so as to hydrolyze the protein sample; (3) and (3) performing mass spectrometry on the hydrolysate obtained in the step (2) by using a high-resolution mass spectrometer. The method has the advantages of simple steps, rapid treatment, less introduced impurities and low cost, and can obtain original protein sequence information by identifying the specific hydrolysis fragments of the protein, and specifically, the bottom-up protein analysis method can be used as a powerful tool for analyzing protein characteristic peptide fragments and fingerprint maps, thereby specifically identifying the target protein.
The method for rapid hydrolysis analysis of protein under high temperature and high pressure according to the above embodiment of the present invention will be described in detail with reference to FIGS. 1-2.
S100, mixing a protein sample with acid to prepare an acidic solution
According to an embodiment of the present invention, an acidic solution is prepared by mixing a protein sample with an acid, wherein the protein sample contains aspartic acid.
According to an embodiment of the invention, the pH value of the acidic solution may be 1-5, and the inventors found that, when the protein is heated in an acidic environment, particularly in a weak acid environment, the C-terminal and N-terminal sites of aspartic acid on the peptide chain of the protein can be cleaved without introducing protease, and if the acidity of the acidic solution is too low, the hydrolysis efficiency of the protein is low, and if the acidity is too high, the protein is likely to undergo other types of structural changes, such as over-hydrolysis at non-specific amino acid sites, and the accuracy of subsequent mass spectrometry is affected. According to the invention, by controlling the pH value of the acidic solution to be 1-5, the hydrolysis efficiency of protein can be obviously improved, and side reactions in the hydrolysis process of the protein can be greatly reduced, so that the analysis result is accurate and reliable. Preferably, the pH value of the acidic solution can be 2-3, so that the side reaction in the protein hydrolysis process can be further reduced, and the accuracy and reliability of the subsequent analysis result can be further improved.
According to another embodiment of the present invention, the kind of the acid in the present invention is not particularly limited, and those skilled in the art can select the acid according to actual needs only to satisfy the condition that the acid can ionize or partially ionize hydrogen ions in water. For example, the acid may be an inorganic acid and/or an organic acid, and for example, the inorganic acid may be hydrogen chloride or the like, and the organic acid may be formic acid or the like.
According to another embodiment of the present invention, the type and form of the protein sample are not particularly limited, and those skilled in the art can select the protein sample according to actual needs, as long as the protein sample contains aspartic acid. For example, the protein sample may be in a powder state or a solution state; the protein sample can be recombinant human ubiquitin protein, bovine serum albumin, recombinant tuberculosis antigen Esat-6 protein, recombinant tuberculosis antigen Cfp-10 protein, ribonuclease or horseradish catalase, etc.
According to another embodiment of the present invention, if the protein sample contains intramolecular disulfide bonds, the protein sample may be mixed with a reducing agent in advance before preparing the acidic solution, so as to cleave the disulfide bonds in the protein sample, thereby further improving the accuracy and reliability of the subsequent analysis results.
S200, heating the acidic solution to a high-temperature high-pressure state in a closed environment to hydrolyze the protein sample
According to an embodiment of the present invention, the acidic solution is heated to a high temperature and high pressure state in a closed environment to hydrolyze the protein sample. The inventor finds that the protein is heated in an acidic environment, and the front and rear sites of aspartic acid on a peptide chain of the protein can be broken without introducing protease, so that macromolecular organic matters such as protease autolysate and the like can be avoided, and non-volatile impurities such as inorganic salts and the like which are not beneficial to mass spectrometric detection in the preparation process of the protease can be avoided; compared with the conventional enzymatic hydrolysis immobilized enzyme technology and the like, the method does not need to carry out pretreatment on a hydrolysis reaction device, and the hydrolysis device is simple to build and has good repeatability; in addition, compared with the conventional enzymolysis temperature of 37 ℃ and the conventional enzymolysis time of 12-24h, the method has the advantages that the protein is hydrolyzed under the high-temperature and high-pressure state, the enzymolysis efficiency is greatly improved, and the time consumption is remarkably reduced.
According to an embodiment of the present invention, referring to fig. 2, the mechanism by which the N-terminal and C-terminal of aspartic acid in a protein sample are specifically cleaved under acidic conditions is: the aspartic acid residue contains free carboxyl, and the free carboxyl reacts with two side peptide bonds (-CO-NH-) adjacent to the aspartic acid under acidic condition to generate five-membered ring/six-membered ring intermediate, and then the peptide bonds are broken to realize the hydrolysis of the protein. Wherein, in FIG. 2, "Asp" in "Asp-X" and "X-Asp" represents aspartic acid, "X" represents an arbitrary amino acid, "R1"and" R2"each represents an amino acid chain.
According to another embodiment of the invention, when the acidic solution is heated in a closed environment, the heating time can be not more than 20min, and the inventor finds that if the heating time is too long, other side reactions such as over-hydrolysis and the like of the protein sample are easily caused, and compared with the conventional enzymolysis time of 12-24h, the invention can heat the acidic solution to a high-temperature high-pressure state in a short time by controlling the heating time to be not more than 20min, and obviously improve the hydrolysis efficiency of the protein on the premise that the protein hydrolysis is caused to occur at front and rear sites of aspartic acid. Preferably, the heating time can be 1-4 min, so that not only can the side reaction of the protein sample be further avoided, but also the hydrolysis efficiency of the protein can be further improved, and the accuracy and reliability of the subsequent analysis result can be ensured.
According to another embodiment of the present invention, the high temperature and high pressure state refers to a subcritical state in which the temperature of the solution is higher than its boiling point under atmospheric pressure, but the liquid state is maintained due to high pressure several times as much as atmospheric pressure generated in the closed space.
According to another embodiment of the present invention, the temperature of the acidic solution under high temperature and high pressure may be 105 to 200 ℃, the pressure may be 1.2 to 16atm (1atm means one standard atmosphere), for example, the temperature can be 105 ℃, 115 ℃, 125 ℃, 135 ℃, 145 ℃, 155 ℃, 165 ℃, 175 ℃, 185 ℃ or 195 ℃, the pressure can be the pressure corresponding to the corresponding temperature, only 1.2 to 16atm is needed, the inventor finds that the hydrolysis efficiency of the protein can be obviously improved by increasing the temperature and the pressure of the acidic solution, however, if the temperature of the acidic solution is too high or the pressure is too high, side reactions such as excessive hydrolysis of the protein sample may also occur, and by controlling the temperature and pressure conditions, the method can effectively avoid side reactions of the protein sample, remarkably improve the hydrolysis efficiency of the protein, and ensure the accuracy and reliability of subsequent analysis results. Preferably, the temperature of the acidic solution under the high-temperature and high-pressure state can be 150-200 ℃, and the pressure can be 5-16 atm, so that the hydrolysis efficiency can be further improved on the premise of effectively avoiding side reactions of the protein sample.
According to another embodiment of the present invention, the acidic solution may be heated in a micro-closed reaction vessel, which may be a micro-capillary reactor or a micro-autoclave. Among them, the micro-closed reaction vessel is preferably capable of supporting an internal pressure of not less than 20 atm.
According to another embodiment of the present invention, the capillary tube in the microcapillary reactor may be a flexible capillary tube or a rigid capillary tube, wherein the flexible capillary tube may be a high molecular polymer capillary tube, such as a polytetrafluoroethylene capillary tube, and the rigid capillary tube may be a stainless steel capillary tube. Further, the capillary tube may be a slender capillary tube, one end of the capillary tube may be connected to a syringe or a syringe pump to absorb the acidic solution, and two ends of the capillary tube may be sealed by pressurization, for example, the high molecular polymer capillary tube may be sealed by radial pressurization and clamping, and the stainless steel capillary tube may be sealed by axial pressurization and plugging.
According to another embodiment of the present invention, when the acidic solution is placed in the micro-closed reaction container for heating, the micro-closed reaction container may be heated by a contact heating method or a non-contact heating method, wherein a heat source of the contact heating method may be an electric heating ceramic sheet or an electric resistance wire, and a heat source of the non-contact heating method may be a fire source or a hot air blower, etc. Preferably, when a non-contact heating method is adopted, a heat conduction material can be filled between the outer wall of the capillary tube and the heat generating unit, so that the heat transfer efficiency can be further improved.
According to still another embodiment of the present invention, the capillary may have an inner diameter of not more than 4mm and a length of not more than 20cm, and preferably, the capillary may have an inner diameter of not more than 2mm and a length of not more than 10 cm. Therefore, the heating efficiency can be further improved, and the time consumption of the whole process flow is greatly shortened.
S300, performing mass spectrometry on the obtained hydrolysate by adopting a high-resolution mass spectrometer
According to the embodiment of the invention, the obtained hydrolysate can be subjected to mass spectrometry by using a high-resolution mass spectrometer, wherein the mass spectrometry is a secondary tandem mass spectrometry.
According to a specific embodiment of the invention, the obtained hydrolysate can be mixed with a polar solvent in advance and then analyzed by a high-resolution mass spectrometer, the hydrolyzed protein has less charges, and the hydrolyzed protein can increase the charges carried by the hydrolyzed protein fragments by mixing the hydrolysate with the polar solvent (such as methanol), so that the signal-to-noise ratio during the preparation of mass spectrometry is remarkably improved, and the structural analysis of the protein sample with high flux and high sensitivity is realized.
According to another embodiment of the invention, the mass concentration of the protein in the hydrolysate is 1-200 ppm, so that the adverse effects of excessive protein content in the hydrolysate on a mass spectrometer and an analysis result can be effectively avoided.
According to another embodiment of the invention, the sample injection mode of mass spectrometry can be a nano-flow electrospray ion source, and the sample injection mode of the electrospray ion source has the characteristics of high flux, high signal-to-noise ratio and the like, so that the detection sensitivity can be remarkably improved, the requirement on the original protein content is reduced, the method is suitable for detection and identification of trace samples, and the structural analysis of high flux and high sensitivity of the protein samples is realized.
The inventor finds that the protein is heated in an acidic environment, and the front and rear sites of aspartic acid on a peptide chain of the protein can be broken without introducing protease, so that macromolecular organic matters such as protease autolysate and the like can be avoided, and non-volatile impurities such as inorganic salts and the like which are not beneficial to mass spectrometric detection in the preparation process of the protease can be avoided; compared with the conventional enzymatic hydrolysis immobilized enzyme technology and the like, the method does not need to carry out pretreatment on a hydrolysis reaction device, and the hydrolysis device is simple to build and has good repeatability; in addition, compared with the conventional enzymolysis temperature of 37 ℃ and the conventional enzymolysis time of 12-24h, the method has the advantages that the protein is hydrolyzed under the high-temperature and high-pressure state, the enzymolysis efficiency is greatly improved, and the time consumption is remarkably reduced. In summary, the rapid hydrolysis analysis method for protein under high temperature and high pressure condition of the embodiments of the present invention not only has simple steps, rapid processing, less introduced impurities and low cost, but also can obtain original protein sequence information by identifying specific hydrolysis fragments of protein, and specifically, the bottom-up protein analysis method can be used as a powerful tool for analyzing protein characteristic peptide fragments and fingerprint, thereby specifically identifying target protein.
In summary, the method for analyzing rapid hydrolysis of protein under high temperature and high pressure according to the above embodiment of the present invention has the following advantages: (1) compared with the proteolysis technology, the method uses the acidic solution as a reaction system, does not need to introduce macromolecular organic matters such as protease and the like, avoids generating macromolecular organic matters such as protease autolysate and the like, and simultaneously avoids introducing inorganic salts and other impurities which are not beneficial to mass spectrum detection in the preparation process of the protease; (2) compared with the conventional enzymolysis temperature of 37 ℃ and the conventional enzymolysis time of 12-24h, the method has the advantages that the enzymolysis efficiency is greatly improved by hydrolyzing the protein under the high-temperature and high-pressure state, and the time consumption is remarkably reduced; (3) compared with the conventional enzymatic hydrolysis immobilized enzyme technology and the like, the method does not need to carry out pretreatment on a hydrolysis reaction device, and the hydrolysis device is simple to build and has good repeatability; (4) the sample introduction mode during mass spectrometry can be a nano-flow electrospray ion source, and compared with the traditional matrix assisted laser dissociation technology, the method can obviously improve the detection sensitivity, reduce the requirement on the original protein content, and is suitable for the detection and identification of trace samples.
According to a second aspect of the present invention, the present invention provides a micro-capillary reactor for use in the above-mentioned rapid hydrolysis analysis method of protein under high temperature and high pressure conditions. According to an embodiment of the present invention, as shown in fig. 3, the micro-capillary reactor includes a heating capillary 100 and a heating controller 200.
Wherein the heating capillary 100 further comprises: the capillary tube 110, the valve, the metal tube 130, the heating wire 140 and the thermistor 160, wherein the valve comprises a first valve 121 and a second valve 122, the first valve 121 and the second valve 122 are respectively arranged at two ends of the capillary tube 110, and the first valve 121 and the second valve 122 control the closed state of the capillary tube 110; the metal tube 130 is sleeved outside the capillary tube 110 and located between the first valve 121 and the second valve 122, and a heat conduction material (not shown) is filled between the inner surface of the metal tube 130 and the outer surface of the capillary tube 110; the heating wire 140 is wound on the outer surface of the metal pipe 130 and the surface of the heating wire 140 is coated with an insulating layer 150; the thermistor 160 is arranged outside the oxygen-insulating layer 150 in the middle of the metal tube 130 and is in direct contact with the metal tube 130 through a small hole on the surface of the insulating layer 150; the heating controller 200 includes a controller 210 and a power amplifier 220, and the controller 210 is connected to the heated capillary tube 100 through the power amplifier 220. When the micro capillary tube reactor is used for heating acid solution, the capillary tube can be used for containing the acid solution, the capillary tube filled with the acid solution is sealed by the first valve and the second valve, the acid solution in the capillary tube is indirectly heated by the heating wires through the metal tube, heat conduction materials are filled between the inner surface of the metal tube and the outer surface of the capillary tube to improve the heat transfer efficiency, the thermistor is used for feeding back the real-time heating temperature, and the power amplifier is used for realizing the starting and stopping of the heating of the capillary tube through a channel of a signal control circuit of the receiving controller. Therefore, the acidic solution can be quickly brought into a high-temperature and high-pressure state, so that the quick hydrolysis of the protein is realized.
According to an embodiment of the present invention, the capillary 110 may be a high molecular polymer capillary, the valve includes a screw and a clamping tube (not shown), the clamping tube has a thread matching with the screw, the clamping tube is sleeved on two ends of the capillary, and the screw is connected to the clamping tube through the thread, if the valve needs to be closed, the screw is rotated to press the hose to block the liquid flow; if the valve is required to be opened, the screw is reversely rotated, and the hose is restored under the action of elastic force to allow the liquid to flow.
According to another embodiment of the present invention, the insulating layer 150 may be an insulating tape, and the insulating tape can effectively perform an auxiliary bonding function at a high temperature, wherein the insulating layer 150 may be divided into an inner layer and an outer layer, the inner layer of the insulating tape is used to isolate the heating wire 140 from the metal tube 130, so as to prevent a short circuit of a system circuit, and the resistance wire can be wound on the metal tube 130 along the inner layer of the insulating tape, so as to enlarge a heating area; the outer layer of insulating tape is used for encapsulating the heating wire 140 and has a certain heat preservation effect.
According to still another embodiment of the present invention, the capillary may have an inner diameter of not more than 4mm and a length of not more than 20cm, and preferably, the capillary may have an inner diameter of not more than 2mm and a length of not more than 10 cm. Therefore, the heating efficiency can be further improved, and the time consumption of the whole process flow is greatly shortened.
According to another embodiment of the present invention, the thermistor 160 can be a glass-encapsulated NTC thermistor, and the resistance thereof can vary from 500K Ω to 5K Ω at 25-200 ℃.
According to another embodiment of the present invention, the central control unit of the controller 210 may be a single chip, and the single chip receives the voltage signal of the thermistor, and controls the power amplifier by modulating the square wave through PWM, and the power amplifier is connected to the heating wire for heating.
According to another embodiment of the present invention, the single chip microcomputer may use ATMega328P and is matched with an Arduino UNO type circuit board. The working voltage of the single chip microcomputer is 5V, and a 7805 type voltage stabilizing tube is adopted inside the single chip microcomputer to stabilize the working voltage. The board card contains an analog sampling port with the precision of 10 bits. And the USB serial port communication protocol is connected with a computer to perform program programming and parameter monitoring. And c language program compiling logic control algorithm is carried out by using open source software Arduino, and the system sampling rate is 10 Hz. And closed loop PI feedback regulation is adopted, the final effect reaches the preset temperature within 25s, and the temperature control precision within +/-3 ℃ within 5 min.
According to another embodiment of the present invention, the power amplifier may include an L298n chip, and the circuit on/off of the output port is controlled by PWM modulation square waveform.
It should be noted that the features and methods described above for the microcapillary reactor are also applicable to the above-mentioned rapid hydrolysis analysis method of protein under high temperature and high pressure.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The general method comprises the following steps:
the method for analyzing the rapid hydrolysis of the protein under the high-temperature and high-pressure state comprises the following specific analysis processes:
(1) the protein sample powder (or solution) to be treated is diluted by adding it to the diluent. If the protein to be treated is in a solution state, the protein to be treated can be blown dry by using nitrogen flow to remove volatile impurities, and then the protein to be treated is added into the diluent for dilution. The diluent is a formic acid-water solution with the volume fraction of 2.5%.
(2) And adding the diluted protein solution obtained in the step into a micro capillary reactor for heating, wherein the preset reaction temperature is 105-200 ℃, and the heating time is not more than 20 min.
(3) And taking out the solution obtained in the step and diluting the solution. The dilution criterion is the concentration of the protein solution suitable for mass spectrometric detection. Calculated as mass fraction of protein, is generally controlled to within 200 ppm. Then adding pure methanol with the same volume as the solution of the analyte to obtain the analyte. The addition of methanol can promote the ionization of polypeptide molecules, improve the ionization efficiency of the electrospray ion source and enhance the signal-to-noise ratio of mass spectra.
Micro capillary reactor
The adopted micro capillary tube reactor is divided into a heating capillary tube and a heating controller.
The heated capillary consists of:
the capillary is used for bearing a reaction system, and the capillary is made of polytetrafluoroethylene material and has a melting point higher than 210 ℃. The capillary has an outer diameter of 1.6mm, an inner diameter of 0.8mm and a length of 30 cm. When the device is in actual use, one end of the capillary tube is connected with the injection needle pump, and the operations of sucking and pushing the solution are performed from the other side.
The valve is used for clamping two ends of the hose to pressurize and block the liquid from flowing through during the reaction. During the heating of the solution, the temperature of the system will rise to 150 ℃, at which time the aqueous solution inside the capillary will be in a state of high temperature and high pressure, and the internal pressure may reach 10 atm. The valve consists of a screw and a clamping tube, wherein the screw is a copper screw with the diameter of 2mm, the clamping tube is a copper tube with the inner diameter of 3mm and provided with a lateral threaded hole with the diameter of 2mm, a flexible capillary tube penetrates through the copper tube and is assembled with the screw, and if the valve needs to be tightly closed, the screw is rotated to press the hose to block the liquid circulation; if the valve is required to be opened, the screw is reversely rotated, and the hose is restored under the action of elastic force to allow the liquid to flow.
The metal pipe is sleeved on the outer side of the capillary pipe so as to enable the capillary pipe to be uniformly heated, and the metal pipe is made of a 304 type stainless steel cylindrical pipe, the outer diameter of the metal pipe is 2.5mm, the inner diameter of the metal pipe is 2mm, and the length of the metal pipe is 75 mm. The high thermal conductivity material is filled in the gap between the metal tube and the flexible capillary tube to make the heat transfer more uniform. The high heat conducting material is heat conducting silicone grease with heat conducting system higher than 4.15W/m.k.
The heating wire is used as a heating material to heat the system and is packaged by an insulating adhesive tape. The insulating tape is made of polyimide materials, the decomposition temperature of the insulating tape is higher than 400 ℃, and the insulating tape can effectively play a role in auxiliary bonding at high temperature. The insulating tape 106 is divided into an inner layer and an outer layer, and the insulating tape of the inner layer is used for isolating the heating wire from the metal pipe and preventing a system circuit from short circuit; a resistance wire with the length of about 20cm is wound on the metal tube along the inner layer of the insulating tape, so that the heating area is enlarged; the outer layer of insulating tape is used for packaging the heating wire and has a certain heat preservation effect. The heating wire is made of Ni80Cr20 type nickel-chromium alloy resistance wire with the diameter of 0.25mm, the resistivity of the resistance wire is 22.21 omega/m, and the total resistance value is 10 omega.
The thermistor is tightly attached to the central position of the metal tube and used for feeding back real-time heating temperature. The thermistor is a glass-packaged negative temperature coefficient thermistor, and the resistance change range of the thermistor is 500-5K omega under the temperature change range of 25-200 ℃.
The heating controller consists of the following parts:
the controller uses a single chip as a central control unit. Specifically, ATMega328P was used, and matched with Arduino UNO type circuit board. The single chip microcomputer of the model has the working voltage of 5V, and a 7805 voltage stabilizing tube is adopted inside the single chip microcomputer to stabilize the working voltage. The board card contains an analog sampling port with the precision of 10 bits. And the USB serial port communication protocol is connected with a computer to perform program programming and parameter monitoring. C language program compiling temperature closed-loop control algorithm is carried out by using open source software, and the system sampling rate is 10 Hz.
The power amplifier contains a model L298n chip. The circuit of the output port of the chip is controlled to be switched on and off by receiving the square wave sent by the controller in a PWM (pulse width modulation) mode. The system is powered by a 12V regulated power supply. The maximum power of the system is 11W, and the idle power is 1W. After the system is started, the preset temperature can be reached within 25s, and the temperature drifts within +/-3 ℃ within 5 min.
Example 1
Rapid specific hydrolysis result analysis of recombinant human ubiquitin protein in weak acid environment
The recombinant human ubiquitin protein is a single-chain protein containing 76 amino acids, and the molecular weight is 8.57 KDa. The protein contains 5 aspartic acid sites, which are bolded in FIG. 4. Can be broken into 6 specific fragments under the condition of weak acid hydrolysis. The assay for this protein was as follows:
(a) and 5.2 mu L of 2mg/ml recombinant human ubiquitin protein solution is taken, 20.8 mu L of formic acid aqueous solution with the volume fraction of 2.5 percent is added, and the mixture is shaken and shaken evenly to obtain 26 mu L of reaction system. In the reaction system, the protein concentration is 0.4mg/mL, the volume fraction of formic acid is 2%, and the pH value before reaction is 2.2;
(b) sucking the reaction system into a micro capillary reactor through an injection pump, presetting a heating temperature of 150 ℃, and heating for 2min under a sealed condition. When the preset heating temperature is reached, the pressure in the tube is not more than 6 atm. Then closing the heating device, cooling and taking out the solution;
(c) adding 26 mu L of methanol into the solution obtained in the step, and fully mixing;
(d) and detecting the solution obtained in the step by using nanoESI-Q-TOF-MS, and analyzing the result.
The mass spectrogram obtained in the above steps is shown in fig. 5, and as can be seen from fig. 5, after specific hydrolysis of ubiquitin protein in a weak acid environment, the ubiquitin protein is broken at Asp-X and X-Asp sites, and 6 specific peptide fragments are newly generated, and the polypeptide fragment peak marked in fig. 4 contains all 6 specific peptide fragments and has a high signal-to-noise ratio. Each peptide fragment is detected by MS/MS secondary spectrogram. Wherein the hydrolysis result of partial polypeptide has a defect of-18 Da (delta m/z) on a mass spectrogram, and water (H) is removed from the inside of the peptide fragment due to local overhigh temperature2O) molecules, which also illustrates that the reaction temperature cannot be raised indefinitely to accelerate the reaction.
Example 2
Rapid specific hydrolysis result analysis of bovine serum albumin in weak acid environment
Bovine serum albumin is a single chain protein containing 607 amino acids and has a molecular weight of about 66.7 kDa. The protein also contains 12 disulfide bonds to maintain the high-order structure of the protein. The protein contains 40 aspartic acid sites, which are bolded in FIG. 6. Can be disintegrated into 36 specific polypeptide fragments under the condition of weak acid hydrolysis. Since bovine serum albumin contains multiple intramolecular disulfide bonds, cleavage is required before the reaction to further perform polypeptide sequence analysis. Therefore, a pretreatment step of disulfide bond cleavage is required in addition to the specific hydrolysis analysis method for the protein. The method comprises the following specific steps:
(a) taking 2.81mg of bovine serum albumin freeze-dried powder, and adding 1mL of water to prepare 2.81mg/mL of protein solution;
(b) and (3) taking 80 mu L of the solution, adding 10 mu L of 60mM dithiothreitol aqueous solution, adding 22.5 mu L of 10% formic acid aqueous solution by volume fraction, shaking up, and taking out 26 mu L to serve as a reaction system. In the reaction system, the protein concentration is 2.00mg/mL, the dithiothreitol concentration is 5.33mM, the formic acid volume fraction is 2%, and the pH value before the reaction is 2.2;
(c) sucking the reaction system into a micro capillary reactor through an injection pump, presetting a heating temperature of 150 ℃, and heating for 5min under a sealed condition. When the preset heating temperature is reached, the pressure in the tube is not more than 6 atm. Then closing the heating device, cooling and taking out the solution;
(d) adding 234 μ L methanol into the solution obtained in the above step, and mixing thoroughly;
(e) and detecting the solution obtained in the step by using nanoESI-Q-TOF-MS, and analyzing the result.
The mass spectrum obtained in the above step is shown in FIG. 7. Wherein dithiothreitol introduced in step (b) is a highly efficient reducing agent, and is commonly used for reducing disulfide bonds in proteins to sulfhydryl groups (-SH). In the traditional protease digestion method, a dithiothreitol solution or other reducing agents are used for cutting off disulfide bonds in protein for pretreatment of the protein, and the common experimental conditions are that the protein is heated in a water bath at 50 ℃ for 15 min-1 h. The analysis method provides that dithiothreitol solution is mixed in a reaction system to directly carry out specific acid hydrolysis reaction, and compared with the traditional sample pretreatment method, the steps are simplified in time.
As can be seen from FIG. 7, the bovine serum albumin undergoes specific hydrolysis in a weak acid environment, and is cleaved at Asp-X and X-Asp sites, resulting in the new generation of 25 specific peptide fragments, which are marked by solid underlines in FIG. 6 and the polypeptide fragment peaks marked in the mass spectrum of FIG. 6. The experimental results cover 53.5% of the amino acid sequence by calculation on FIG. 6. The method has good cutting effect on long-chain polypeptides such as bovine serum albumin and can be used for identifying proteins with large mass numbers.
Example 3
And (3) analyzing the result of rapid specific hydrolysis of the recombinant tuberculosis antigen Esat-6 protein in a weak acid environment.
The tubercle bacillus is a pathogenic source of tuberculosis, and the Esat-6 and Cfp-10 proteins secreted by the tubercle bacillus are effective means for determining whether organisms are pathogenic or not.
The recombinant tuberculosis antigen Esat-6 protein is a single-chain protein containing 95 amino acids, a tag modification at the end part is introduced in a production link, and the molecular weight is 11.1 kDa. The protein contains 2 aspartic acid sites, which are bolded in FIG. 8. Can be broken into 3 specific fragments under the condition of weak acid hydrolysis. The assay for this protein was as follows:
(a) 5.2 mu L of 1.50mg/ml recombinant tuberculosis antigen Esat-6 protein solution is taken, 20.8 mu L of formic acid aqueous solution with volume fraction of 2.5 percent is added, and shaking are carried out to obtain 26 mu L of reaction system. In the reaction system, the protein concentration is 0.30mg/mL, the volume fraction of formic acid is 2%, and the pH value before reaction is 2.2;
(b) sucking the reaction system into a micro capillary reactor through an injection pump, presetting a heating temperature of 150 ℃, and heating for 2min under a sealed condition. When the preset heating temperature is reached, the pressure in the tube is not more than 6 atm. Then closing the heating device, cooling and taking out the solution;
(c) adding 26 mu L of methanol into the solution obtained in the step, and fully mixing;
(d) and detecting the solution obtained in the step by using nanoESI-Q-TOF-MS, and analyzing the result.
The mass spectrogram obtained in the above step is shown in fig. 9, and as can be seen from fig. 9, Esat-6 protein is subjected to specific hydrolysis in a weak acid environment, and is broken at Asp-X and X-Asp sites, so that 3 specific peptide fragments are newly generated, and the polypeptide fragment peaks marked in fig. 9 contain all 3 specific peptide fragment peaks and corresponding metal ion binding peaks thereof, and have a high signal-to-noise ratio. Each peptide fragment is detected by MS/MS secondary spectrogram. Wherein the hydrolysis result of the #2 polypeptide has a defect of-18 Da at delta m/z on a mass spectrogram, and water is removed from the inside of a peptide segment (H) due to local overhigh temperature2O) molecule.
Example 4
And (3) analyzing the result of rapid specific hydrolysis of the recombinant tuberculosis antigen Cfp-10 protein in a weak acid environment.
The recombinant tuberculosis antigen Cfp-10 protein is a single-chain protein containing 100 amino acids, a tag modification at the end part is introduced in a production link, and the molecular weight is 11.7 kDa. The protein contains 5 aspartic acid sites, which are bolded in FIG. 10. Can be broken into 6 specific fragments under the condition of weak acid hydrolysis. The assay for this protein was as follows:
(1) 5.2 mu L of recombinant tuberculosis antigen Esat-6 protein solution of 2.70mg/ml is taken, 20.8 mu L of formic acid aqueous solution with volume fraction of 2.5 percent is added, and shaking is carried out to obtain 26 mu L of reaction system. In the reaction system, the protein concentration is 0.54mg/mL, the volume fraction of formic acid is 2%, and the pH value before reaction is 2.2;
(2) sucking the reaction system into a micro capillary reactor through an injection pump, presetting a heating temperature of 150 ℃, and heating for 2min under a sealed condition. When the preset heating temperature is reached, the pressure in the tube is not more than 6 atm. Then closing the heating device, cooling and taking out the solution;
(3) adding 52 mu L of methanol into the solution obtained in the step, and fully mixing;
(4) and detecting the solution obtained in the step by using nanoESI-Q-TOF-MS, and analyzing the result.
The mass spectrogram obtained by the steps is shown in fig. 11, as can be seen from fig. 11, the Cfp-10 protein is subjected to specific hydrolysis in a weak acid environment, and is broken at Asp-X and X-Asp sites, so that 4 specific peptide fragments are newly generated, and the polypeptide fragment peaks marked in fig. 11 contain 4 specific peptide fragment peaks in the middle of a peptide chain and have high signal-to-noise ratio. Each peptide fragment is detected by MS/MS secondary spectrogram.
Example 5
The difference from example 1 is that:
(b) sucking the reaction system into a micro capillary reactor through an injection pump, presetting the heating temperature to 200 ℃, heating for 2min under a sealed condition, controlling the pressure to be 16atm, and taking out the solution;
the mass spectrum obtained in the above step is shown in FIG. 12. In fig. 12, fragment peptide fragments #6 and #4 obtained by specific hydrolysis of ubiquitin proteins are labeled with P1 and P2, respectively, and multiple charge peaks of original ubiquitin proteins 10+, 9+ and 8+ are labeled with S1, S2 and S3. By comparing the relative intensities of the peaks at the corresponding positions in fig. 5, it can be seen that the P1, P2 peaks in fig. 12 have higher intensity ratios than the S1, S2, S3 peaks. The following conclusions can thus be drawn: compared with example 1, the hydrolysis efficiency of ubiquitin protein is higher when the heating temperature is 200 ℃.
Example 6
The difference from example 1 is that:
(b) sucking the reaction system into a micro capillary reactor through an injection pump, presetting a heating temperature of 105 ℃, heating for 8min under a sealed condition, controlling the pressure to be 1.2atm, and taking out the solution;
the mass spectrum obtained in the above step is shown in FIG. 13. In fig. 13, fragment peptide fragments #6 and #4 obtained by specific hydrolysis of ubiquitin proteins are labeled with P1 and P2, respectively, and multiple charge peaks of original ubiquitin proteins 10+, 9+ and 8+ are labeled with S1, S2 and S3. By comparing the intensity ratios of the P1, P2 peaks to the S1, S2, S3 peaks at the corresponding positions of fig. 5 and fig. 13, it can be seen that: compared with example 1, specific hydrolysis of protein occurred at a heating temperature of 105 ℃, but the hydrolysis efficiency was poor. By heating for a longer time, the protein already begins to appear partial hydrolysis fragments, such as P1 labeled unilaterally specific fragment peptide segment # 6 of ubiquitin. However, the hydrolysis efficiency was low under these conditions, and the P2-labeled fragment peptide # 4 position did not produce significant fragmentation results. The multi-charge peaks marked by S1, S2 and S3 as the original ubiquitin proteins 10+, 9+ and 8+ are shown as the peaks with the highest relative ionic strength in the mass spectrogram.
Comparative example 1
The difference from example 1 is that:
(b) sucking the reaction system into a micro capillary reactor through an injection pump, presetting the heating temperature to 50 ℃, heating for 2min under a sealed condition, controlling the pressure to be 1atm, and taking out the solution;
the mass spectrum obtained in the above step is shown in FIG. 14. In fig. 14, fragment peptide fragments #6 and #4 obtained by specific hydrolysis of ubiquitin proteins are labeled with P1 and P2, respectively, and multiple charge peaks of original ubiquitin proteins 10+, 9+ and 8+ are labeled with S1, S2 and S3. It can be seen that the original protein did not dissociate specifically significantly under the pyrolysis conditions of this comparative example. This indicates that lower temperatures are not conducive to specific hydrolysis of the protein.
Comparative example 2
The difference from example 1 is that:
(b) sucking the reaction system into a micro capillary reactor through an injection pump, presetting a heating temperature of 230 ℃, heating for 2min under a sealed condition, controlling the pressure to be 28atm, and taking out the solution;
the mass spectrum obtained in the above step is shown in FIG. 15. In fig. 15, fragment peptide fragments #6 and #4 obtained by specific hydrolysis of ubiquitin proteins are labeled with P1 and P2, respectively, and multiple charge peaks of original ubiquitin proteins 10+, 9+ and 8+ are labeled with S1, S2 and S3. Compared with the results of example 5, the relative intensities of the P1 and P2 peaks are further improved compared with the S1, S2 and S3, and the reaction efficiency of the system is further improved compared with that of fig. 12. However, when the heating temperature is raised to 230 ℃, the service life of the reactor, especially the micro capillary made of polytetrafluoroethylene, is affected by the excessive temperature and pressure. Therefore, the device is not suitable for working at 230 ℃ for a long time, and the heating temperature is controlled to be 30 ℃ below the softening point of the polytetrafluoroethylene material, so that the safety and reproducibility of the heating device are ensured.
In combination with the above examples of protein applications, the present invention provides a novel and rapid specific detection method for protein identification by using the rapid specific hydrolysis analysis method under weak acid environment. The method has good compatibility for proteins with different sources and different lengths, and can be used for detecting protein markers of diseases. Compared with the traditional protease digestion technology, the method greatly shortens the reaction time; the introduction of impurities such as protease autolysate, high-concentration salt environment required by enzyme digestion and the like is avoided, the detection can be carried out by a nanoflow spray ion source without separation and enrichment means, and the signal-to-noise ratio of a reaction product detection result is improved.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise. In the present invention, unless otherwise expressly stated or limited, the terms "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integral to one another; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. A method for rapid hydrolysis analysis of protein under high temperature and high pressure, comprising:
(1) mixing a protein sample with acid to prepare an acidic solution, wherein the protein sample contains aspartic acid;
(2) heating the acidic solution to a high temperature and high pressure state in a closed environment so as to hydrolyze the protein sample;
(3) performing mass spectrometry on the hydrolysate obtained in the step (2) by adopting a high-resolution mass spectrometer,
wherein, in the step (1), the protein sample containing intramolecular disulfide bonds is mixed with a reducing agent and acid to prepare the acidic solution, the reducing agent is used for cutting the disulfide bonds in the proteins, and then the acidic solution is directly subjected to the operation of the step (2);
in the step (2), the temperature of the acidic solution is 105-150 ℃ in a high-temperature and high-pressure state;
in the step (2), the acid solution is placed in a micro closed reaction container to be heated, and the micro closed reaction container is a micro capillary reactor; the micro capillary reactor comprises a heating capillary tube and a heating controller, the heating capillary tube further comprises a capillary tube and a valve, the valve comprises a first valve and a second valve, the first valve and the second valve are respectively arranged at two ends of the capillary tube, the first valve and the second valve control the closed state of the capillary tube, the capillary tube is a slender capillary tube, and one end of the capillary tube is connected with an injector or an injection pump to absorb the acidic solution;
in the step (3), the hydrolysate obtained in the step (2) is mixed with a polar solvent in advance and then is analyzed by a high-resolution mass spectrometer.
2. The analytical method according to claim 1, wherein in step (1), the pH value of the acidic solution is 1 to 5, preferably 2 to 3;
optionally, the protein sample is recombinant human ubiquitin protein, bovine serum albumin, recombinant tuberculosis antigen Esat-6 protein, recombinant tuberculosis antigen Cfp-10 protein, ribonuclease or horseradish catalase.
3. The analytical method according to claim 1 or 2, wherein in step (2), the heating time is not more than 20min, preferably 1 to 4 min;
optionally, in the step (2), the pressure of the acidic solution in the high-temperature and high-pressure state is 1.2-6 atm.
4. The analysis method according to claim 3, wherein in the step (3), the mass concentration of the protein in the hydrolysate is 1-200 ppm;
optionally, in the step (3), the sample introduction manner of the mass spectrometry is a nano-flow electrospray ion source.
5. The analytical method of claim 1, wherein in the microcapillary reactor, the capillary is made of a flexible capillary or a rigid capillary;
optionally, the capillary tube has an inner diameter of no greater than 4mm and a length of no greater than 20 cm; preferably, the capillary tube has an inner diameter of no more than 2mm and a length of no more than 10 cm.
6. The analytical method of claim 5, wherein the capillary is a high molecular polymer capillary, and the capillary is sealed by radial pressure clamping; alternatively, the first and second electrodes may be,
the capillary is a stainless steel capillary, and the capillary is sealed in an axial pressurizing and plugging mode.
7. The analytical method according to claim 6, wherein in the step (2), the heating is performed by a contact heating method or a non-contact heating method;
optionally, the heating is performed by a non-contact heating method, and a heat conduction material is filled between the outer wall of the capillary tube and the heat generating unit.
8. A microcapillary reactor for use in the analytical method according to any one of claims 1 to 7, wherein the microcapillary reactor comprises:
a heated capillary, the heated capillary further comprising:
a capillary tube;
the valves comprise a first valve and a second valve, the first valve and the second valve are respectively arranged at two ends of the capillary tube, and the first valve and the second valve control the closed state of the capillary tube;
the metal pipe is sleeved outside the capillary and positioned between the first valve and the second valve, and heat conduction materials are filled between the inner surface of the metal pipe and the outer surface of the capillary;
the heating wire is wound on the outer surface of the metal pipe, and an insulating layer is coated on the surface of the heating wire;
the thermistor is arranged in the middle of the metal tube and is in contact with the metal tube through a small hole in the surface of the insulating layer;
the heating controller comprises a controller and a power amplifier, and the controller is connected with the heating capillary tube through the power amplifier.
9. The micro capillary reactor according to claim 8, wherein the capillary is a high molecular polymer capillary, the valve comprises a screw and a clamping tube, the clamping tube has a thread matching with the screw, the clamping tube is sleeved at two ends of the capillary, and the screw is connected with the clamping tube through the thread.
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