A method for determining the concentration of TNF-alpha based on peptide segment isotope dilution mass spectrometry

Abstract

The application belongs to the technical field of protein standard substance value determination, and particularly relates to a TNF-alpha value determination method based on a peptide segment isotope dilution mass spectrometry. The application discloses a TNF-alpha value determination method based on a peptide segment isotope dilution mass spectrometry, which comprises characteristic peptide segment selection (determining ANALLANGVELR and VNLLSAIK as the characteristic peptide segments for quantification according to the amino acid sequence of TNF-alpha), characteristic peptide segment purity determination, characteristic peptide segment mass spectrometry parameter optimization, characteristic peptide segment linear range determination, TNF-alpha protein sample pretreatment, sample enzyme cutting solution mass spectrometry analysis, and characteristic peptide segment content calculation. Finally, the concentration of the target protein in the solution is calculated according to the molecular weights of TNF-alpha and the characteristic peptide segments, and is taken as the quantification result. The TNF-alpha specificity determined by the method is high, and the peptide segments can be traced to the national amino acid standard substance, and finally traced to the SI unit system, so that the accuracy and reliability of the quantification result are ensured.

Description

Technical Field

[0001] This invention belongs to the field of protein standard substance determination technology, specifically relating to a TNF-α determination method based on peptide isotope dilution mass spectrometry. Background Technology

[0002] Tumor necrosis factor-α (TNF-α) is the most potent anti-tumor cytokine discovered to date, possessing a wide range of biological activities. TNF-α can promote the production and secretion of IL-1, IL-2, and IL-6 through the secretion of activated T cells and macrophages, inducing inflammatory responses and promoting the expression of IL-2R, EGFR, and major histocompatibility antigen class Ag, forming a complex immune network and playing a crucial role in the host's defense response. Studies have found that TNF-α acts as a mediator in Crohn's disease, rheumatoid arthritis, psoriasis, and tumor proliferation and differentiation, as well as inhibiting apoptosis. Therefore, accurate measurement of TNF-α is crucial for early screening and prognosis in disease treatment.

[0003] Isotope dilution mass spectrometry (IDMS) is a method for determining substances using stable isotopes. A certain amount of isotopically labeled compound is added to the sample, and after thorough mixing, it is hydrolyzed or enzymatically digested. The ratio of labeled to unlabeled substances is then calculated using mass spectrometry. This is one of the main techniques for quantifying protein standards. However, commonly used Isotope dilution mass spectrometry cannot distinguish the source of amino acids, requires very high purity of the raw materials, and lacks specificity. Therefore, it is necessary to develop new, more specific quantitative methods to supplement the detection of TNF-α content.

[0004] Accurate measurement of TNF-α is crucial for early screening and prognosis of the disease. TNF-α detection methods are mainly divided into bioactivity assays, immunological assays, and molecular biological assays, but all have significant drawbacks. For example, the MTT assay, widely used in bioactivity assays, has poor specificity and is easily affected by sample interference; immunological assays suffer from cross-reactivity and sensitivity is highly dependent on antibodies; molecular biological methods detect cytokine mRNA and cannot objectively reflect protein expression levels, leading to discrepancies with other methods. Clinical testing commonly uses a wide variety of immunoassay reagents, and results from different products from different manufacturers can vary considerably. To ensure the traceability, accuracy, and comparability of measurement results and to achieve standardization in clinical testing, it is necessary to establish accurate and reliable reference methods to meet the needs of clinical testing. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to design a technical solution for TNF-α determination based on peptide isotope dilution mass spectrometry.

[0006] The present invention is implemented using the following technical solutions: The first aspect of this invention provides a method for quantifying TNF-α based on peptide isotope dilution mass spectrometry, comprising the following steps: S.1 Characteristic peptide selection: Based on the TNF-α amino acid sequence, the two peptides ANALLANGVELR and VNLLSAIK were identified as quantitative characteristic peptides; S.2 Synthesis of Characteristic Peptides and Isotope-Labeled Peptides: Synthesis of characteristic peptides ANALLANGVELR and VNLLSAIK and two isotope-labeled peptides ANALLA ( 13 C3, 15 N)NGVELR, VNLLSA( 13 C3, 15 N)IK; S.3 Determination of purity of characteristic peptides: Using leucine and valine as standards, and isotopically labeled leucine, valine or isoleucine as internal standards, the two synthesized unlabeled characteristic peptides were accurately quantified by amino acid isotope dilution mass spectrometry. S.4 Optimization and analysis of mass spectrometry parameters for characteristic peptides: The parent ion and daughter ion of the characteristic peptides and isotope-labeled peptides were determined, and the collision energy was adjusted to select the ion with the strongest signal as the quantitative ion pair. The mass spectrometry parameters were then optimized. The characteristic peptides were analyzed by mass spectrometry using a liquid chromatography-tandem mass spectrometry system, and the mass spectrometry acquisition adopted the multiple reaction monitoring mode. S.5 Determination of the linear range of characteristic peptides: Measure the TNF-α protein solution to be tested, add and weigh the corresponding volume of isotope internal standard solution; calculate the mass of the two target peptides obtained after enzyme digestion based on the amount of TNF-α protein contained in the solution and the purity of the two peptides, prepare standard peptides and isotope internal standard peptides of corresponding concentrations, and make a standard curve. S.6 Pretreatment of TNF-α protein samples for analysis: optimization of enzyme digestion conditions and trypsin degradation; S.7 Calculate the content of characteristic peptides after enzyme digestion, and then calculate the concentration of TNF-α in the solution based on the content as the quantitative result.

[0007] Furthermore, the purity of the characteristic peptide and the isotope-labeled peptide described in step S.2 is required to be above 98%.

[0008] Further, the characteristic peptides described in step S.4 are selected from the following multiple reaction monitoring ion pairs: ANALLANGVELR: (620.7→758.5); VNLLSAIK: (429.1→644.5); IS-ANALLANGVELR: (623.7→762.5); IS-VNLLSAIK: (431.4→648.5).

[0009] Further, the mass spectrometry analysis conditions described in step S.4 are as follows: mobile phase: phase A 0.1% formic acid aqueous solution, phase B 0.1% formic acid acetonitrile solution; program: 0 min: 95% A: 5% B; 4 min: 65% A: 35% B; 6 min: 0% A: 100% B; 7-10 min: 95% A: 5% B; injection volume: 2 μL; flow rate: 0.3 mL·min -1 Mass spectrometry uses a positive ion method for detection and multiple reaction monitoring (MRM) for scanning.

[0010] Further, the preparation of the standard curve in step S.5 specifically includes: accurately weighing the stock solutions of the characteristic peptide and its internal standard peptide, preparing a standard curve working solution with a concentration gradient according to the mass ratio, mixing and performing detection and analysis, establishing a standard curve with the peak area ratio of the characteristic peptide to the isotope-labeled peptide as the x-axis and the mass ratio of the characteristic peptide to its internal standard peptide as the y-axis, and calculating the linearity of the standard peptide in the concentration range of 0.21 μM-8.02 μM, R0. 2 >0.99.

[0011] Furthermore, the concentration of the characteristic peptide of the TNF-α protein to be tested described in step S.5 is within the linear range.

[0012] Furthermore, in step S.6, the enzymatic hydrolysis time is 2-72 h.

[0013] Furthermore, the formula for calculating the content of the characteristic peptides mentioned in step S.7 is shown in formula (2), and the formula for calculating the quantitative result of TNF-α is shown in formula (1):

[0014] In the formula,

[0015] The present invention has the following beneficial effects: Unlike existing technologies that quantify TNF-α based on bioactivity, this invention uses a peptide isotope dilution mass spectrometry method to determine the content of non-standard peptides. Therefore, the TNF-α measured by this method has high specificity, and the peptides can be traced back to national amino acid standard materials, and finally to the SI unit system, ensuring the accuracy and reliability of the quantitative results.

[0016] This method is used to quantify two characteristic peptides: ANALLANGVELR and VNLLSAIK. It can be applied to the isotope dilution mass spectrometry quantification of all TNF-α and related drugs containing these characteristic peptides, such as human TNF-α and injectable recombinant modified human tumor necrosis factor. Attached Figure Description

[0017] Figure 1 To optimize enzyme digestion conditions; Figure 2 The retention peaks of characteristic peptides after enzymatic hydrolysis in Waters ACQUITY UPLC I-Class / Xevo TQ-XS are obtained through multiple reaction monitoring chromatography. Detailed Implementation

[0018] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0019] Example 1: Quantification of His-tagged TNF-α protein using peptide isotope dilution mass spectrometry 1) Selection of characteristic peptides Under the action of trypsin, TNF-α breaks at its specific cleavage site, forming multiple characteristic peptides. By collecting the signals from the enzymatic digests and comparing and matching them with peptide signals in a database, accurate identification of the target protein can be achieved. After multiple batches of enzymatic digestion experiments, the system screened out two characteristic peptides that met the following criteria: good reproducibility, high mass spectrometry detection sensitivity, complete preservation of the cleavage site, suitable sequence length, and inclusion of at least one stable isotope label.

[0020] The peptides ANALLANGVELR and VNLLSAIK were selected as characteristic peptides for His-tagged TNF-α quantification based on peptide isotope dilution mass spectrometry, and their sequences are shown in SEQ ID NO.1: TPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALASHHHHHHH.

[0021] 2) Synthesize characteristic peptides for His-tagged TNF-α protein quantification Two characteristic peptides and their isotope-labeled peptides were obtained by chemical synthesis. The synthesized peptides had an HPLC purity of greater than 98%.

[0022] 3) Accurate measurement of the purity of characteristic peptides Accurately weigh the characteristic peptide sample and amino acid mixed internal standard working solution into an ampoule. After using a vacuum centrifuge to concentrate, add the hydrolysate, purge the air from the ampoule with nitrogen, seal the ampoule with an oxyhydrogen flame sealer, and place it in a metal bath at 110°C for reaction. After drying with nitrogen using a nitrogen evaporator, redissolve the solution, filter it, and place it in a sample vial for testing.

[0023] The high-performance liquid chromatography (HPLC) and mass spectrometry (MS) parameters are shown in the table below. The monitored ion pair parameters are as follows: leucine quantitative ion pair: m / z 132.09 > 86.16; leucine internal standard quantitative ion pair: m / z 139.03 > 92.15; valine quantitative ion pair: m / z 117.97 > 72.06; valine internal standard quantitative ion pair: m / z 123.97 > 77.07.

[0024] Table 1 High Performance Liquid Chromatography Parameters

[0025] Table 2 Mass Spectrometry Parameters

[0026] A high-performance liquid chromatography-isotope dilution mass spectrometry (HPLC-IOSD-MS) method was established using national amino acid standard reference materials to determine the content of stable amino acids in the hydrolysate, and the purity of characteristic peptides was calculated accordingly. For the characteristic peptides ANALLANGVELR and VNLLSAIK, leucine and valine were used for quantification, respectively.

[0027] Table 3. Quantitative results of standard peptides

[0028] 4) Optimization of mass spectrometry parameters for characteristic peptides The ion pairs for characteristic peptide mass spectrometry analysis need to be determined through optimization of characteristic peptide mass spectrometry parameters: (1) Analyze the parent and daughter ions of labeled and unlabeled characteristic peptides, and select the pair with the strongest signal by adjusting the collision energy. The following multiple reaction monitoring ion pairs were selected: ANALLANGVELR: (620.7→758.5); VNLLSAIK: (429.1→644.5); IS-ANALLANGVELR: (623.7→762.5); IS-VNLLSAIK: (431.4→648.5).

[0029] (2) Optimize the mass spectrometry parameters such as cone voltage and fragmentation energy of quantitative ion pairs to make their mass spectrometry response the strongest.

[0030] 5) Liquid chromatography-mass spectrometry analysis of characteristic peptides Characteristic peptides were analyzed by tandem liquid chromatography-mass spectrometry (LC-MS / MS). Mobile phase: Phase A - 0.1% formic acid aqueous solution; Phase B - 0.1% formic acid-acetonitrile solution. Program: 0 min: 95%A: 5%B; 4 min: 65%A: 35%B; 6 min: 0%A: 100%B; 7-10 min: 95%A: 5%B; Injection volume: 2 μL; Flow rate: 0.3 mL / min. -1 Mass spectrometry employs a positive ion method for detection, and the scanning mode is multiple reaction monitoring (MRM). Figure 2 ).

[0031] 6) Determination of the linear range of characteristic peptides Two characteristic peptides were prepared into solutions separately. The synthesized labeled peptide solutions were thoroughly mixed with standard peptides of various concentrations and analyzed. A standard curve was established with the peak area ratio of the characteristic peptide to the isotopically labeled peptide as the x-axis and the mass ratio of the characteristic peptide to its internal standard peptide as the y-axis. The linearity of the standard peptides in the concentration range of 0.21 μM–8.02 μM was calculated, and R0 was determined. 2 >0.999.

[0032] Table 4. Linear Relationship of Standard Peptides

[0033] 7) Prepare His-tagged TNF-α protein solution and standard peptide solution with internal standard added. Accurately weigh the volume of His-tagged TNF-α protein solution to be tested, and add and weigh the corresponding volume of isotope internal standard solution. Based on the amount of TNF-α protein contained in the solution, calculate the mass of the two target peptides obtained after enzyme digestion according to the purity of the two peptides, and prepare standard peptides and isotope internal standard peptides of corresponding concentrations.

[0034] 8) Optimization of enzyme digestion conditions for His-tagged TNF-α protein Accurately weigh a certain mass of human TNF-α sample and its characteristic peptide internal standard working solution, add protein-dissolving surfactant (Rapigest SF) to a final concentration of 0.1%, and induce protein denaturation at 60℃ for 30 min. After equilibration at room temperature, perform disulfide bond modification stepwise: first, reduce with 10 mM DTT solution at 56℃ for 45 min, then perform light-protected alkylation with 50 mM iodoacetamide (IAM) for 30 min, and finally add DTT solution to make the molar ratio of DTT to IAM ≥ 1:1. During the enzymatic digestion process, add trypsin at an enzyme / substrate ratio of 1:50, set 7 time gradients (2-72 h), and perform enzymatic digestion at 37℃. At the reaction termination stage, add trifluoroacetic acid (TFA) to a final concentration of 0.5%, and continue treatment at 37℃ for 50 min to fully inactivate enzyme activity. Finally, centrifuge at 13,000×g for 10 min to obtain the supernatant enzyme digest. Take out the supernatant, dilute with 0.1% formic acid-water solution, desalt by column chromatography, evaporate to dryness, reconstitute, filter, and then analyze. Ultimately, 36 hours was chosen as the optimal enzymatic digestion time, and trypsin was added to the digestion system every 12 hours. Figure 1 ).

[0035] 9) Calculate the content of characteristic peptides after enzyme digestion, and then calculate the concentration of His-tagged TNF-α in the solution based on the content as the quantitative result.

[0036]

[0037] In the formula,

[0038] The concentration of His-tagged TNF-α protein in solution was calculated based on the molecular weight of the His-tagged TNF-α protein and the molecular weight of its characteristic peptides, serving as the quantitative result. The quantitative results of His-tagged TNF-α protein using isotope dilution mass spectrometry based on peptide analysis are shown in the table below.

[0039] Table 5. Results of His-tagged TNF-α protein content determination (mg / g) by isotope dilution mass spectrometry based on peptide analysis.

[0040] Example 2: Quantification of humanized TNF-α protein based on peptide isotope dilution mass spectrometry 1) Selection of characteristic peptides Under the action of trypsin, TNF-α breaks at its specific cleavage site, forming multiple characteristic peptides. By collecting the signals from the enzymatic digests and comparing and matching them with peptide signals in a database, accurate identification of the target protein can be achieved. After multiple batches of enzymatic digestion experiments, the system screened out two characteristic peptides that met the following criteria: good reproducibility, high mass spectrometry detection sensitivity, complete preservation of the cleavage site, suitable sequence length, and inclusion of at least one stable isotope label.

[0041] The peptides ANALLANGVELR and VNLLSAIK were selected as characteristic peptides for the quantification of humanized TNF-α using peptide isotope dilution mass spectrometry, and their sequences are shown in SEQ ID NO.2. VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL.

[0042] 2) Synthesize characteristic peptides for quantification of humanized TNF-α protein. Two characteristic peptides and their isotope-labeled peptides were obtained by chemical synthesis. The synthesized peptides had an HPLC purity of greater than 98%.

[0043] 3) Accurate measurement of the purity of characteristic peptides Accurately weigh the characteristic peptide sample and amino acid mixed internal standard working solution into an ampoule. After using a vacuum centrifuge to concentrate, add the hydrolysate, purge the air from the ampoule with nitrogen, seal the ampoule with an oxyhydrogen flame sealer, and place it in a metal bath at 110°C for reaction. After drying with nitrogen using a nitrogen evaporator, redissolve the solution, filter it, and place it in a sample vial for testing.

[0044] The high-performance liquid chromatography (HPLC) and mass spectrometry (MS) parameters are shown in Tables 1 and 2. The monitored ion pair parameters are as follows: leucine quantitative ion pair: m / z 132.09 > 86.16; leucine internal standard quantitative ion pair: m / z 139.03 > 92.15; valine quantitative ion pair: m / z 117.97 > 72.06; valine internal standard quantitative ion pair: m / z 123.97 > 77.07. An HPLC-isotope dilution mass spectrometry method was established using national amino acid standard reference materials to determine the stable amino acid content in the hydrolysate, and the purity of characteristic peptides was calculated accordingly. For the characteristic peptides ANALLANGVELR and VNLLSAIK, leucine and valine were used for quantification, respectively, and the results are shown in Table 3.

[0045] 4) Optimization of mass spectrometry parameters for characteristic peptides The ion pairs for characteristic peptide mass spectrometry analysis need to be determined through optimization of characteristic peptide mass spectrometry parameters: (1) Analyze the parent and daughter ions of labeled and unlabeled characteristic peptides, and select the pair with the strongest signal by adjusting the collision energy. The following multiple reaction monitoring ion pairs were selected: ANALLANGVELR: (620.7→758.5); VNLLSAIK: (429.1→644.5); IS-ANALLANGVELR: (623.7→762.5); IS-VNLLSAIK: (431.4→648.5).

[0046] (2) Optimize the mass spectrometry parameters such as cone voltage and fragmentation energy of quantitative ion pairs to make their mass spectrometry response the strongest.

[0047] 5) Liquid chromatography-mass spectrometry analysis of characteristic peptides Characteristic peptides were analyzed by tandem liquid chromatography-mass spectrometry (LC-MS / MS). Mobile phase: Phase A - 0.1% formic acid aqueous solution; Phase B - 0.1% formic acid-acetonitrile solution. Program: 0 min: 95%A: 5%B; 4 min: 65%A: 35%B; 6 min: 0%A: 100%B; 7-10 min: 95%A: 5%B; Injection volume: 2 μL; Flow rate: 0.3 mL / min. -1 Mass spectrometry was performed using a positive ion method with multiple reaction monitoring (MRM) scanning.

[0048] 6) Determination of the linear range of characteristic peptides Two characteristic peptides were prepared into solutions separately. The synthesized labeled peptide solutions were thoroughly mixed with standard peptides of various concentrations and analyzed. A standard curve was established with the peak area ratio of the characteristic peptide to the isotopically labeled peptide as the x-axis and the mass ratio of the characteristic peptide to its internal standard peptide as the y-axis. The linearity of the standard peptides in the concentration range of 0.21 μM–8.02 μM was calculated, and R0 was determined. 2 >0.999. The linear relationship of the standard peptides is shown in Table 4.

[0049] 7) Prepare humanized TNF-α protein solution and standard peptide solution with added internal standard. Accurately weigh the volume of humanized TNF-α protein solution to be tested, and add and weigh the corresponding volume of isotope internal standard solution. Based on the amount of TNF-α protein contained in the solution, calculate the mass of the two target peptides obtained after enzyme digestion according to the purity of the two peptides, and prepare standard peptides and isotope internal standard peptides of corresponding concentrations.

[0050] 8) Optimization of enzymatic digestion conditions for humanized TNF-α protein Accurately weigh a certain mass of human TNF-α sample and its characteristic peptide internal standard working solution, add protein-dissolving surfactant (Rapigest SF) to a final concentration of 0.1%, and induce protein denaturation at 60℃ for 30 min. After equilibration at room temperature, perform disulfide bond modification stepwise: first, reduce with 10 mM DTT solution at 56℃ for 45 min, then perform light-protected alkylation with 50 mM iodoacetamide (IAM) for 30 min, and finally add DTT solution to make the molar ratio of DTT to IAM ≥ 1:1. During the enzymatic digestion process, add trypsin at an enzyme / substrate ratio of 1:50, set 7 time gradients (2-72 h), and perform enzymatic digestion at 37℃. At the reaction termination stage, add trifluoroacetic acid (TFA) to a final concentration of 0.5%, and continue treatment at 37℃ for 50 min to fully inactivate enzyme activity. Finally, centrifuge at 13,000×g for 10 min to obtain the supernatant enzyme digest. Take out the supernatant, dilute with 0.1% formic acid-water solution, desalt by column chromatography, evaporate to dryness, reconstitute, filter, and then analyze. Ultimately, 36 h was selected as the optimal enzymatic digestion time, and trypsin was added to the digestion system every 12 h.

[0051] 9) Calculate the content of characteristic peptides after enzyme digestion, and then calculate the concentration of humanized TNF-α in the solution based on the content as the quantitative result.

[0052]

[0053] In the formula,

[0054] The concentration of humanized TNF-α protein in solution was calculated based on the molecular weight of the humanized TNF-α protein and the molecular weight of its characteristic peptides, serving as the quantitative result. The quantitative results of humanized TNF-α protein using isotope dilution mass spectrometry based on peptide analysis are shown in the table below.

[0055] Table 6. Results of determination of humanized TNF-α protein content (μg / g) by isotope dilution mass spectrometry based on peptide analysis.

Claims

1. A method for quantifying TNF-α based on peptide isotope dilution mass spectrometry, characterized in that, Includes the following steps: S.1 Characteristic peptide selection: Based on the TNF-α amino acid sequence, the two peptides ANALLANGVELR and VNLLSAIK were identified as quantitative characteristic peptides; S.2 Synthesis of Characteristic Peptides and Isotope-Labeled Peptides: Synthesis of characteristic peptides ANALLANGVELR and VNLLSAIK and two isotope-labeled peptides ANALLA ( 13 C3, 15 N)NGVELR, VNLLSA( 13 C3, 15 N)IK; S.3 Determination of purity of characteristic peptides: Using leucine and valine as standards, and isotopically labeled leucine, valine or isoleucine as internal standards, the two synthesized unlabeled characteristic peptides were accurately quantified by amino acid isotope dilution mass spectrometry. S.4 Optimization and analysis of mass spectrometry parameters for characteristic peptides: The parent ion and daughter ion of the characteristic peptides and isotope-labeled peptides were determined, and the collision energy was adjusted to select the ion with the strongest signal as the quantitative ion pair. The mass spectrometry parameters were then optimized. The characteristic peptides were analyzed by mass spectrometry using a liquid chromatography-tandem mass spectrometry system, and the mass spectrometry acquisition adopted the multiple reaction monitoring mode. S.5 Determination of the linear range of characteristic peptides: Measure the TNF-α protein solution to be tested, add and weigh the corresponding volume of isotope internal standard solution; calculate the mass of the two target peptides obtained after enzyme digestion based on the amount of TNF-α protein contained in the solution and the purity of the two peptides, prepare standard peptides and isotope internal standard peptides of corresponding concentrations, and make a standard curve. S.6 Pretreatment of TNF-α protein samples for analysis: optimization of enzyme digestion conditions and trypsin degradation; S.7 Calculate the content of characteristic peptides after enzyme digestion, and then calculate the concentration of TNF-α in the solution based on the content as the quantitative result.

2. The method as described in claim 1, characterized in that, The purity of the characteristic peptide and the isotope-labeled peptide mentioned in step S.2 must be above 98%.

3. The method as described in claim 1, characterized in that, The characteristic peptides described in step S.4 are selected from the following multiple reaction monitoring ion pairs: ANALLANGVELR: (620.7→758.5); VNLLSAIK: (429.1→644.5); IS-ANALLANGVELR: (623.7→762.5); IS-VNLLSAIK: (431.4→648.5).

4. The method as described in claim 1, characterized in that, The mass spectrometry analysis conditions described in step S.4 are as follows: mobile phase: phase A 0.1% formic acid aqueous solution, phase B 0.1% formic acid acetonitrile solution; program: 0 min: 95% A: 5% B; 4 min: 65% A: 35% B; 6 min: 0% A: 100% B; 7-10 min: 95% A: 5% B; injection volume: 2 μL; flow rate: 0.3 mL·min -1 Mass spectrometry uses a positive ion method for detection and multiple reaction monitoring (MRM) for scanning.

5. The method as described in claim 1, characterized in that, Step S.5, the preparation of the standard curve, specifically includes: accurately weighing the stock solutions of the characteristic peptide and its internal standard peptide, preparing a standard curve working solution with a concentration gradient according to the mass ratio, mixing thoroughly, and performing detection and analysis. A standard curve is established by plotting the peak area ratio of the characteristic peptide to the isotope-labeled peptide on the x-axis and the mass ratio of the characteristic peptide to its internal standard peptide on the y-axis. The linearity of the standard peptide in the concentration range of 0.21 μM-8.02 μM is calculated, R0. 2 >0.

99.

6. The method as described in claim 1, characterized in that, The concentrations of the characteristic peptides of the TNF-α protein to be tested, as described in step S.5, are within the linear range.

7. The method as described in claim 1, characterized in that, In step S.6, the enzymatic hydrolysis time is 2-72 h.

8. The method according to claim 1, characterized in that, The formula for calculating the content of the characteristic peptides in step S.7 is shown in formula (2), and the formula for calculating the quantitative result of TNF-α is shown in formula (1): In the formula,

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