Pretreatment method of protein sample and peptide diagram analysis method

Abstract

The invention is applicable to the technical field of biology, and provides a pretreatment method of a protein sample and a peptide diagram analysis method, which comprises the following steps: (a) carrying out a heating denaturation reduction reaction on the protein sample in the presence of a denaturing agent and a reducing agent, and then adding an alkylation reagent to carry out a dark alkylation reaction; (b) filling the reaction liquid into a dialysis bag, and carrying out multiple times of liquid change dialysis; (c) concentrating the dialyzed sample solution, and adding a protease solution to carry out enzymolysis reaction; (d) adding an acid solution into the solution after enzymolysis to terminate the reaction, centrifuging and taking supernate, the method overcomes the problem of loss of a membrane adsorption sample caused by a traditional ultrafiltration method, is particularly suitable for pretreatment of a trace protein sample, reduces the use amount of the sample and the experiment cost, can be compatible with synchronous digestion reaction of multiple enzymes, and has a wide application prospect. The pretreated sample is analyzed by LC-MS / MS, a chromatogram with good peak shape and good separation degree can be obtained, and reliable data is provided for quality control and structural identification of protein drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for pretreatment of protein samples and a method for peptide mapping analysis. Background Technology

[0002] Peptide mapping analysis is a core technology in the biopharmaceutical field for sequence confirmation, post-translational modification analysis, and quality control of protein drugs. The accuracy of its analytical results is highly dependent on whether the target protein and its enzymatically digested peptides can be completely and accurately preserved during the sample pretreatment stage.

[0003] Currently, mainstream pretreatment methods generally rely on ultrafiltration-based centrifugation for desalting and buffer replacement. However, the non-specific adsorption of proteins and peptides onto ultrafiltration membrane materials (such as polyethersulfone and regenerated cellulose) is an inherent and intractable drawback. This adsorption leads to: Significant sample loss: For trace amounts (<500 μg) or highly hydrophobic precious protein samples, the recovery rate can be as low as 50%, making subsequent analysis impossible or data distorted.

[0004] Introducing uncontrollable biases: Adsorption is not homogeneous, and peptides with different properties have different adsorption rates, causing mass spectrometry results to fail to accurately reflect the original proportion of peptides in the sample, which is particularly detrimental to quantitative analysis.

[0005] High cost: To compensate for the losses, the starting sample size had to be increased, which led to a sharp increase in experimental costs for candidate drug proteins, which are extremely expensive per milligram.

[0006] Although the industry has attempted to alleviate this problem by developing "low-adsorption" filter membranes and adding carrier proteins or surfactants, these are all temporary solutions: low-adsorption membranes can only reduce rather than eliminate adsorption; and additives can contaminate samples and interfere with subsequent chromatographic and mass spectrometric analyses.

[0007] Other techniques, such as solid-phase extraction, are cumbersome and have inconsistent packing material properties; trichloroacetic acid precipitation is prone to incomplete protein denaturation and precipitation, and is not suitable for trace samples.

[0008] Therefore, a pressing technical challenge has long existed in this field: how can a sample pretreatment method completely avoid membrane adsorption, and provide a high recovery rate and high fidelity solution, especially for peptide mapping analysis of trace protein samples? The present invention is proposed to overcome this technical bottleneck. Summary of the Invention

[0009] This invention provides a protein sample pretreatment method and a peptide mapping analysis method, aiming to overcome the shortcomings of the prior art and provide a protein pretreatment method and peptide mapping analysis method with low sample loss and good reproducibility.

[0010] This invention is implemented as follows: a method for pretreatment of protein samples, comprising the following steps: (a) Denaturation-reduction alkylation treatment: The protein sample was subjected to a denaturation-reduction reaction by heating in the presence of a denaturing agent and a reducing agent, followed by the addition of an alkylating agent for a dark alkylation reaction to obtain a reaction solution; (b) Dialysis desalination: The reaction solution obtained in step (a) is placed into a dialysis bag and dialyzed multiple times at 4°C using phosphate buffer to remove small molecule impurities and salts; (c) Concentration and enzymatic hydrolysis: The dialyzed sample solution is concentrated to a predetermined volume, dispensed, and then a protease solution is added for enzymatic hydrolysis. (d) Termination and preparation: Add acid solution to the enzymatically digested solution to terminate the reaction, centrifuge and take the supernatant to obtain the peptide sample to be analyzed.

[0011] Preferably, in step (a), the denaturing agent is guanidine hydrochloride with a final concentration of 4-8 M; the reducing agent is dithiothreitol with a final concentration of 10-30 mM; the alkylating agent is iodoacetamide with a final concentration of 40-60 mM; the temperature of the heating denaturation-reduction reaction is 50-60 °C, and the time is 20-40 min; the time of the dark alkylation reaction is 20-40 min.

[0012] Preferably, in step (b), the molecular weight cutoff of the dialysis bag is 5-9 kDa; the pH of the phosphate buffer is 7.0-8.0 and the concentration is 5-20 mM; the number of dialysis sessions is 2-4, and the total time is 12-36 hours.

[0013] Preferably, in step (c), the concentration is carried out by centrifugation at a speed of 10,000-15,000 rpm; the protease includes at least one of trypsin and chymotrypsin; the enzyme-to-protein ratio of the enzymatic hydrolysis reaction is 1:40-60; when trypsin is used, the hydrolysis temperature is 36-38°C and the reaction time is 3-5 hours; when chymotrypsin is used, the hydrolysis temperature is 23-27°C and the reaction time is 3-5 hours.

[0014] Preferably, in step (d), the acid solution is a formic acid solution or a trifluoroacetic acid solution with a final concentration of 0.5%-2%; the centrifugation speed is 10000-15000 rpm and the time is 3-10 min.

[0015] This invention also provides a method for peptide mapping analysis of protein samples, comprising the following steps: The protein samples were pretreated using the above method to obtain peptide samples to be analyzed. The peptide samples to be analyzed were separated and detected using a high-performance liquid chromatography-tandem mass spectrometry system. In the liquid chromatography, mobile phase A is an aqueous solution containing 0.05%-0.2% formic acid, and mobile phase B is an acetonitrile solution containing 0.05%-0.2% formic acid, using a gradient elution program; The specific testing steps are as follows: Instrument preparation: Turn on the column oven of the HPLC equipment, first rinse the column (rinse with 10% mobile phase A2 and 90% mobile phase B2 for 20 min, then rinse with 90% mobile phase A2 and 10% mobile phase B2 for 20 min), then equilibrate the column with the initial ratio (97% mobile phase A1 and 3% mobile phase B1) for 20 min until the baseline is stable. Sample analysis: Then, the sample is injected and analyzed according to the chromatographic conditions; Result determination: If the chromatogram of the sample to be tested has good separation, uniform peak distribution, and clear characteristics, and meets the expectations of enzymatic hydrolysis, then the sample to be tested is qualified; otherwise, it is unqualified.

[0016] Preferably, the liquid chromatography uses a C18 column, a column temperature of 45-55℃, a flow rate of 0.15-0.25 mL / min, an injection volume of 20 μL, and a run time of 100 min. Preferably, the gradient elution procedure is as follows: From 0 to 2 minutes, the volume ratio of mobile phase A to mobile phase B was 97:3. From 2 to 85 minutes, the volume fraction of mobile phase B increased linearly from 3% to 32%. Within 85-90 minutes, the volume fraction of mobile phase B increased linearly from 32% to 90%. For 90-95 minutes, the volume fraction of mobile phase B is maintained at 90%. 95.1–100 min, the volume fraction of mobile phase B rapidly decreased from 90% to 3% and remained thereuntil the end. Preferably, the mass spectrometry detection uses an electrospray ionization source, positive ion mode scanning, and a scanning range of 300-2000.

[0017] Compared with the prior art, the embodiments of this application have the following main advantages: Completely eliminating membrane adsorption and achieving ultra-high sample recovery rates: This invention creatively uses dialysis technology to replace ultrafiltration centrifugation, completely avoiding sample contact with the filter membrane, fundamentally solving the problem of non-specific adsorption of proteins and peptides. Especially for trace protein samples (100 μg level), the recovery rate can be stably maintained at about 73%, providing a sufficient material basis for subsequent analysis.

[0018] Ensuring the authenticity and reliability of analytical data: By avoiding selective adsorption, the prepared peptide mixture fully retains the compositional information of the original sample, resulting in high peak capacity, full peak shape, and uniform distribution in the final chromatogram. This ensures higher peptide sequence coverage and more sensitive detection of minor modifications such as oxidation and deamidation, greatly improving the accuracy and reliability of peptide chromatogram analysis results.

[0019] Excellent compatibility and flexibility: The procedure of this invention is compatible with the individual or simultaneous enzymatic digestion of various proteases (such as trypsin and chymotrypsin), meeting different analytical needs. Furthermore, the method is robust, reproducible, and easy to implement in different laboratories.

[0020] Reduced overall experimental costs: Although dialysis is time-consuming, its consumable costs are far lower than those of high-performance ultrafiltration centrifuge tubes. More importantly, the extremely high recovery rate avoids the waste of repeated experiments due to insufficient samples, significantly reducing the overall cost of a single successful analysis, especially for the development of expensive protein drugs. Attached Figure Description

[0021] Figure 1 This is a flowchart of a protein sample pretreatment method provided by the present invention.

[0022] Figure 2 This is a chromatogram of the sample solution digested with Trypsin in the sample to be tested in Example 1.

[0023] Figure 3 This is a chromatogram of the sample solution digested with Chymotrypsin in the sample to be tested in Example 1.

[0024] Figure 4 This is a chromatogram of the sample solution digested with Trypsin in the test sample of Comparative Example 1.

[0025] Figure 5 The image shows the chromatogram of the sample solution digested with Chymotrypsin in the test sample of Comparative Example 1.

[0026] Figure 6 This is the chromatogram of the blank reference standard in Example 1.

[0027] Figure 7 This is the chromatogram of the standard in Example 1.

[0028] Figure 8 The image shows the chromatogram of the experimental sample in Example 1. Detailed Implementation

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0030] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0031] Example 1 This invention provides a method for protein sample pretreatment and peptide mapping analysis. 1. Instruments and equipment: Three-in-one liquid chromatography-mass spectrometry (LC-MS) system: Orbitrap Eclipse, Thermo Fisher High-speed centrifuge: Centrifuge 5810R, Eppendorf Ultrasonic Cleaner: SB-5200DT, Ningbo Xinzhi Heated metal bath: DC-100, Youning Medical refrigerator / freezer: YCD-EL519, Meiling pH meter: C227410434, Mettler Toledo Instruments Electronic balance: 3142811472, Sartorius Centrifugal concentrator: 7310038, LABCONCO 2. Experimental reagents: Trypsin: V5111, Promega Chymotrypsin: V1061, Promega Dithiothreitol (DTT): D9163-5G, Sigma Iodoacetamide (IAM): I6125-10G, Sigma 8M Guanidine Hydrochloride: G7294-100mL, Sigma Tris(hydroxymethyl)aminomethane (Tris): 252859-100G, Sigma Formic acid FA: 85178, Thermo Fisher Acetonitrile ACN: 51101, Thermo Fisher Disodium hydrogen phosphate Na2HPO4•12H2O: (Source: Guoyao) Sodium dihydrogen phosphate (NaH2PO4•2H2O): (Source: China National Pharmaceutical Group) Concentrated hydrochloric acid (HCl): Chinese medicine Sodium chloride (NaCl): (This appears to be a product name or label, possibly related to a Chinese medicine company.) 3. Experimental consumables: Accucore™ C18 HPLC column: 17126-152130, Thermo Fisher Screw cap: 6PSC9ST1, Thermo Fisher Screw cap micro sample vials: 6ESV9-04PP, Thermo Fisher 0.2mL EP tube: PCR-02-C, Axygen 1.5mL EP tube: MVT-150-C, Axygen Dialysis bag: MC25-7, GPCSCI 2. Experimental Procedure 1) Sample preparation: Take the sample to be tested and dissolve or dilute it with ultrapure water to 1 mg / mL for later use; 2) Enzyme preparation: Take Trypsin and Chymotrypsin powder, dissolve them in ultrapure water to a concentration of 1 mg / mL, aliquot into EP tubes, 1 μL / tube, and store at -20℃ for later use. 3) Reagent preparation: 1.2114 g of Tris was dissolved in 10 mL of ultrapure water and the pH was adjusted to 7.8 with concentrated hydrochloric acid. 1 mol / L DTT, weigh 15.4 mg DTT and dissolve it in 0.1 mL of ultrapure water; 18.5 mg of 1 mol / L IAM is dissolved in 0.1 mL of ultrapure water; For 50mM NaCl + 10mM PB (phosphate buffer), weigh 0.584g NaCl, 1.16g Na2HPO4•12H2O, and 0.224g NaH2PO4•2H2O, and bring the volume to 2L with ultrapure water, adjusting the pH to 7.5; for 10mM PB, weigh 2.32g Na2HPO4•12H2O and 0.448g NaH2PO4•2H2O, and bring the volume to 4L with ultrapure water, adjusting the pH to 7.5. 10% FA (formic acid) solution: Take 10 μL of FA into an EP tube, add 90 μL of ultrapure water, mix well and set aside. 4) Preparation of the mobile phase: For mobile phase A1, take 1 mL of FA and add it to 1 L of H2O. Sonicate for 30 min and set aside. For mobile phase B1, take 1 mL of FA and add it to 1 L of ACN. Sonicate for 30 min and set aside. Mobile phase A2, 1L H2O, sonicated for 30 minutes and then ready for use; In mobile phase B2, in 1L of ACN, sonicate for 30 minutes and then set aside. 5) Sample processing, such as Figure 1 As shown, the steps are as follows: Take 100 μg of the sample to be tested into a 1.5 mL clean EP tube (and simultaneously prepare standard controls: blank (ddH2O) and standard (commercially purchased trastuzumab (Herceptin) with a final concentration of 0.70 μg / μL and a sample recovery rate of approximately 70%) as controls), add 8 M guanidine hydrochloride (final concentration to 6 M) and 1 M Tris-HCl (final concentration to 50 mM), for a total volume of 500 μL; Add 1M DTT (final concentration to 20mM), incubate at 57℃ for 30min, cool to room temperature, then add 1M IAM (final concentration to 50mM), and let stand in the dark for 30min. Transfer the sample solution from the 1.5 mL EP tube to a dialysis bag (7 kDa MWCO), immerse it in dialysis buffer (50 mM NaCl + 10 mM PB, 2 L), dialyze at 4 °C for 2 h and replace the dialysis buffer (10 mM PB, 2 L), dialyze at 4 °C for 8 h and replace the dialysis buffer (10 mM PB, 2 L), and incubate at 4 °C overnight. After dialysis, the sample solution was transferred to a clean EP tube and concentrated to 100 μL using a centrifugal concentrator (centrifugal force 1725 rpm, temperature 37℃, time 30 min). After concentration, the sample solution was dispensed into two clean 0.2 mL EP tubes, 50 μL / tube for a total of two tubes. Add 1 μL of Trypsin and Chymotrypsin enzyme solutions at an enzyme-to-base ratio of 1:50, mix well, react Trypsin at 37℃ for 4 h and Chymotrypsin at 25℃ for 4 h, then add 5 μL of 10% FA solution to terminate the reaction, mix well and centrifuge, take 50 μL of supernatant into a liquid chromatography vial for analysis. 3. Chromatographic conditions Column: Accucore TM C18 (150×2.1mm, 2.6μm); Mobile phase A: Water (containing 0.1% FA); Mobile phase B: Acetonitrile (containing 0.1% FA); Column temperature: 50℃; Flow rate: 0.2 mL / min; Injection volume: 20 μL; Gradient elution of the mobile phase is shown in Table 1 below: Table 1 Gradient elution of the target protein in the mobile phase 4. Mass spectrometry conditions Ion source: Ion Max NG; Ionization mode: HESI; Sheath gas temperature: 320℃; Sheath gas flow rate: 35Arb; Spray voltage: Static; Capillary temperature: 320℃; S-Lens RF Level: 50%; Scan mode: Orbitrap; First-level scan range (m / z): 200-2000; Resolution: 120000; AGC target: 300; Maximum injection time: 100ms; Secondary fragmentation: HCD; Fragmentation energy: 30%; Secondary resolution: 30000; AGC target: 100; Maximum injection time: 200ms.

[0032] 5. Results and Analysis The results are as follows Figure 2 and Figure 3 As shown in the figure, both enzyme digestions were quite thorough, with abundant and uniformly distributed peptide peaks in the chromatogram, indicating good separation. This demonstrates that the pretreatment method of this invention can effectively prepare samples for high-quality peptide mapping analysis.

[0033] Using blank (ddH2O) and standard (Herceptin, final concentration 0.70 ug / ul, sample recovery approximately 70%) as controls, the following results were obtained. Figure 6 , Figure 7 , Figure 8 Spectral analysis revealed that the chromatograms of both the experimental samples and standards showed good separation, uniform peak distribution, and clear characteristics, consistent with the expectations of enzymatic hydrolysis. Furthermore, the samples treated by dialysis exhibited better proteolytic hydrolysis, spectral signal intensity, ion peak abundance, and separation than those treated by ultrafiltration, indicating that the pretreatment method of this invention can effectively prepare samples for high-quality peptide mapping analysis.

[0034] Comparative Example 1 (Protein sample pretreatment using ultrafiltration) Instruments, consumables, and reagents: 10kd ultrafiltration tube: UFC501096 Sigma; Ultraviolet spectrophotometer: Nano Drop One Thermo; Solution I: 7.5 mL 8M guanidine hydrochloride + 0.5 mL 1M Tris-HCl + 2 mL ddH2O, pH adjusted to 7.8; Solution H: 0.06g urea + 2mL 1M Tris-HCl + 18mL ddH2O, pH adjusted to 7.8; Urea: National Pharmaceutical Group Add 100 µg of sample to a 10 kD ultrafiltration centrifuge tube, bring the volume to 500 µl with Solution I, centrifuge at 14000 rpm for 10 min, repeat twice, ensuring the final volume is less than or equal to 100 µl. Add 2 µl of 1 M DTT to the denatured sample, mix well, and incubate at 56 °C for 30 min. Then, remove the sample and cool it to room temperature. Add 52 µl of 1 M IAM to the sample, mix well, and incubate at room temperature in the dark for 30 min. Bring the volume to 500 µl with Solution H, centrifuge at 14000 rpm for 10 min, repeat twice, and finally bring the final volume to 100 µl with Solution H. Aliquot the sample solution into a clean 0.2 mL container. Two EP tubes (50 μL each) were used. 1 μL of Trypsin and 1 μL of Chymotrypsin enzyme solution were added to each tube at an enzyme-to-solvent ratio of 1:50. After mixing, Trypsin was reacted at 37°C for 4 hours, and Chymotrypsin at 25°C for 4 hours. The reaction was then terminated by adding 5 μL of 10% FA solution. After mixing thoroughly, the mixture was centrifuged, and 50 μL of the supernatant was transferred to a liquid chromatography vial for analysis. Figure 4 , Figure 5 .

[0035] The final sample concentration obtained by ultrafiltration was 0.42 ug / μL, with a sample recovery rate of approximately 42%. The final sample concentration obtained by dialysis was 0.73 ug / μL, with a sample recovery rate of approximately 73%.

[0036] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0037] It should be understood that the disclosed apparatus can be implemented in other ways, as illustrated in the embodiments provided in this application. For example, the apparatus embodiments described above are merely illustrative; the division of units described above is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or communication connections shown or discussed may be through some interfaces; the indirect coupling or communication connections between devices or units may be telecommunications or other forms.

[0038] The units described above as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.

Claims

1. A method for pretreatment of protein samples, characterized in that, Includes the following steps: (a) Denaturation-reduction alkylation treatment: The protein sample was subjected to a denaturation-reduction reaction by heating in the presence of a denaturing agent and a reducing agent, followed by the addition of an alkylating agent for a dark alkylation reaction to obtain a reaction solution; (b) Dialysis desalination: The reaction solution obtained in step (a) is placed into a dialysis bag and dialyzed multiple times at 4°C using phosphate buffer to remove small molecule impurities and salts; (c) Concentration and enzymatic hydrolysis: The dialyzed sample solution is concentrated to a predetermined volume, dispensed, and then a protease solution is added for enzymatic hydrolysis. (d) Termination and preparation: Add acid solution to the enzymatically digested solution to terminate the reaction, centrifuge and take the supernatant to obtain the peptide sample to be analyzed.

2. The protein sample pretreatment method as described in claim 1, characterized in that, In step (a), the denaturing agent is guanidine hydrochloride with a final concentration of 4-8 M; the reducing agent is dithiothreitol with a final concentration of 10-30 mM; the alkylating agent is iodoacetamide with a final concentration of 40-60 mM; the temperature of the heating denaturation-reduction reaction is 50-60 °C for 20-40 min; and the time of the dark alkylation reaction is 20-40 min.

3. The protein sample pretreatment method as described in claim 1, characterized in that, In step (b), the molecular weight cutoff of the dialysis bag is 5-9 kDa; the pH of the phosphate buffer is 7.0-8.0 and the concentration is 5-20 mM; the number of dialysis sessions is 2-4, and the total time is 12-36 hours.

4. The protein sample pretreatment method as described in claim 1, characterized in that, In step (c), the concentration is carried out by centrifugation at a speed of 10,000-15,000 rpm; the protease includes at least one of trypsin and chymotrypsin; and the enzyme-to-protein ratio of the enzymatic hydrolysis reaction is 1:40-60.

5. The protein sample pretreatment method as described in claim 4, characterized in that, When using trypsin, the enzymatic hydrolysis temperature is 36-38℃ and the reaction time is 3-5 hours; when using chymotrypsin, the enzymatic hydrolysis temperature is 23-27℃ and the reaction time is 3-5 hours.

6. The protein sample pretreatment method as described in claim 1, characterized in that, In step (d), the acid solution is a formic acid solution or a trifluoroacetic acid solution with a final concentration of 0.5%-2%; the centrifugation speed is 10000-15000 rpm and the time is 3-10 min.

7. A method for peptide mapping analysis of protein samples, characterized in that, Includes the following steps: The protein sample is pretreated using the method described in any one of claims 1-6 to obtain the peptide sample to be analyzed. The peptide samples to be analyzed were separated and detected using a high-performance liquid chromatography-tandem mass spectrometry system. In the liquid chromatography, mobile phase A is an aqueous solution containing 0.05%-0.2% formic acid, and mobile phase B is an acetonitrile solution containing 0.05%-0.2% formic acid, using a gradient elution program.

8. The peptide mapping analysis method for protein samples as described in claim 7, characterized in that, The liquid chromatography used a C18 column with a column temperature of 45-55℃, a flow rate of 0.15-0.25 mL / min, an injection volume of 20 μL, and a run time of 100 min.

9. The peptide mapping analysis method for protein samples as described in claim 7, characterized in that, The gradient elution procedure is as follows: From 0 to 2 minutes, the volume ratio of mobile phase A to mobile phase B was 97:

3. From 2 to 85 minutes, the volume fraction of mobile phase B increased linearly from 3% to 32%. Within 85-90 minutes, the volume fraction of mobile phase B increased linearly from 32% to 90%. For 90-95 minutes, the volume fraction of mobile phase B is maintained at 90%. 95.1-100 min, the volume fraction of mobile phase B rapidly decreased from 90% to 3% and remained thereuntil the end.

10. The peptide mapping analysis method for protein samples as described in claim 7, characterized in that, The mass spectrometry detection uses an electrospray ionization source, positive ion mode scanning, and a scanning range of 300-2000.

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