Fibrinogen quantification method based on characteristic peptide fragment isotope dilution mass spectrometry

Abstract

The invention discloses a fibrinogen quantification method based on a characteristic peptide fragment isotope dilution mass spectrometry. The method comprises the following steps: (1) selection of characteristic peptide fragments: selecting three peptide fragments of VR, DK and YR as the characteristic peptide fragments; (2) chemically synthesizing three characteristic peptide fragments and corresponding isotope labeled peptide fragments; (3) determining the purity of the three characteristic peptide fragments; (4) carrying out enzyme digestion on fibrinogen (FIB) through trypsin; and (5) performing quantitative analysis on the FIB by adopting isotope dilution mass spectrometry of peptide fragments. According to the method, the FIB is valued by using a characteristic peptide fragment isotope dilution mass spectrometry, and the principle is that the concentration of the protein is calculated according to the concentration of the characteristic peptide fragment. According to the method, three characteristic peptide fragments of the FIB are obtained and are not easily influenced by interference components, the concentration of the FIB is calculated by using the three peptide fragments, and the value is determined by using the average value of three results, so that the accuracy of a quantitative result is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein standard material valuation, and in particular to a fibrinogen quantification method based on characteristic peptide segment isotope dilution mass spectrometry. Background Art

[0002] Fibrinogen (FIB) is a glycoprotein synthesized by the liver that plays an important role in blood coagulation and is a precursor to fibrin. It contains 2964 amino acids, has a molecular weight of approximately 340 kDa, and is composed of two identical components (Aα, Bβ, and γ polypeptide chains) forming a hexamer, connected by disulfide bonds. Fibrinogen is coagulation factor I, the most abundant coagulation factor in plasma, and the final coagulation factor in the coagulation cascade. When the body is damaged by certain factors, the concentration of fibrinogen increases significantly by several times. Accurate measurement of plasma fibrinogen helps determine the prognosis of the disease. Plasma fibrinogen is a sensitive biochemical marker that provides an objective scientific basis for clinical treatment selection and prognosis.

[0003] The fibrinogen reference materials currently used in my country have low accuracy in their characteristic quantity determination methods and a single concentration. Furthermore, existing reference materials cannot achieve absolute quantification of protein content. Therefore, there is an urgent need to develop reference materials with accurate measurement values ​​and traceability.

[0004] Amino acid isotope dilution mass spectrometry is a commonly used method for determining protein content, but this method cannot distinguish between amino acids coming from proteins or impurities. This method has high requirements for protein purity and its determination results may be biased high.

[0005] Therefore, developing a method for determining the absolute quantification of fibrinogen and traceable measurement results to SI units has become one of the technical problems that urgently need to be solved in this field. Summary of the Invention

[0006] The present invention aims to provide a fibrinogen quantification method based on isotope dilution mass spectrometry of a characteristic peptide. This method uses isotope dilution mass spectrometry to determine the value of a peptide specific for fibrinogen, resulting in high specificity and accuracy, and the measurement results are traceable to the SI unit of kg.

[0007] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:

[0008] A fibrinogen quantification method based on characteristic peptide isotope dilution mass spectrometry comprises the following steps:

[0009] Step (1) Selection of characteristic peptides: VR, DK, and YR are selected as characteristic peptides, and the sequences of VR, DK, and YR are VTSGSTTTTR, DNENVVNEYSSELEK, and YEASILTHDSSIR, respectively;

[0010] Step (2) Synthesis of characteristic peptides: Synthesize characteristic peptides VR, DK, YR and three isotope-labeled peptides, the sequences of the three isotope-labeled peptides are VTSGSTTTTR (13C6, 15N4), DNENVVNEYSSELEK (13C6, 15N2), and YEASILTHDSSIR (13C6, 15N4).

[0011] Step (3) Accurate quantification of the purity of the characteristic peptide segment: the synthesized peptide segment is hydrolyzed into amino acids, isotope-labeled amino acids are quantitatively added, and the amino acid isotope dilution mass spectrometry method is established using amino acid standard substances as standards to determine the content of stable amino acids in the hydrolyzate, and the purity of the characteristic peptide segment is calculated accordingly;

[0012] Step (4) fibrinogen (FIB) is enzymatically cleaved by trypsin;

[0013] Step (5) using peptide isotope dilution mass spectrometry to accurately quantify the three characteristic peptides in the enzymatic hydrolysate;

[0014] Step (6) Optimization of liquid phase conditions and mass spectrometry conditions for three characteristic peptides: determining the parent ions and daughter ions of the characteristic peptides and isotope-labeled peptides, and optimizing the mass spectrometry parameters;

[0015] Step (7) calculates the contents of the three characteristic peptides in the enzymatic hydrolysate respectively, and calculates the concentration of FIB based on the contents of the characteristic peptides as the fixed value result of the protein.

[0016] Wherein, in the step (3), the peptide VR is quantified by isotope dilution mass spectrometry of valine and arginine, DK is quantified by valine and leucine, and YR is quantified by isoleucine and leucine.

[0017] Wherein, in step (4), the enzymatic hydrolysis time is 16 h, and the ratio of sample to trypsin is 10:1.

[0018] Wherein, in the step (5), the content of the synthesized characteristic peptide segment is calculated using the following formula:

[0019]

[0020] Among them, C AA : concentration of amino acids in solution;

[0021] P: purity of amino acid standards;

[0022] m标 : The mass of the labeled amino acid added to the peptide, mg;

[0023] R 样 : Amino acid chromatographic peak area ratio in the sample, non-labeled / labeled;

[0024] I1: actual mass ratio of low-labeled amino acids, non-labeled / labeled;

[0025] I2: actual mass ratio of high-standard amino acids, non-labeled / labeled;

[0026] R1: low-label amino acid peak area ratio, non-labeled / labeled;

[0027] R2: peak area ratio of high-labeled amino acids, non-labeled / labeled;

[0028] M: mass of VR, DK, and YR sample solutions in parallel samples, mg;

[0029]

[0030] Where Ci represents the characteristic peptide concentration calculated for each amino acid, CAA represents the concentration of the measured amino acid, Mi represents the relative molecular mass of the characteristic peptide, MAA represents the relative molecular mass of each amino acid, and n represents the number of corresponding amino acids in VR, DK, and YR.

[0031] Wherein, in said step (6), the chromatographic column used is ACQUITYUPLC Peptide BEH C18, 2.1 mm × 150 mm, 1.7 μm;

[0032] Mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, the flow rate was 0.2 ml / min, and the injection volume was 10 μL;

[0033] Flow gradient: 0-15 min, 5%-50% B; 15-22 min, 50%-90% B; 22-25 min, 90%-5% B; 25-30 min, 5% B;

[0034] VR 1010.6 / 966.5, DK 885.0 / 572.2, YR 746.6 / 928.6, 13C-VR 1020.5 / 976.5, 13C-DK 889.0 / 572.1, 13C-YR 751.2 / 938.7; and DP and CE values ​​were optimized.

[0035] Wherein, the step (7) is specifically as follows:

[0036] 1) Calculate the mass of the three characteristic peptides after enzyme digestion based on their purity, and prepare the corresponding concentrations of the characteristic peptides and isotope-labeled peptides;

[0037] 2) The three characteristic peptide isotope internal standard solutions were prepared into a mixed solution with a mass ratio of 1:1 to the peptide in the sample solution; the ratios of the peptide standard solution to the isotope internal standard were 0.9:1, 1:1, and 1.1:1;

[0038] 3) Weigh the volume of FIB protein solution to be tested, calculate the mass of the peptide in the FIB sample solution, and add a 1:1 mass ratio of the isotope internal standard mixed solution. The concentration of the characteristic peptide in the FIB to be tested should be in the middle of the determined linear range.

[0039] 4) Calculate the concentration of FIB using the following formula:

[0040]

[0041] Among them, C i : Concentrations of VR, DK, and YR in the solution;

[0042] P: purity of characteristic peptide;

[0043] m 标 : The mass of the labeled peptide added to the characteristic peptide, mg;

[0044] R 样 : chromatographic peak area ratio of characteristic peptides in the sample, non-labeled / labeled;

[0045] I1: actual mass ratio of low-label peptide, non-labeled / labeled;

[0046] I2: actual mass ratio of high-labeled peptides, non-labeled / labeled;

[0047] R1: low-label peptide peak area ratio, non-labeled / labeled;

[0048] R2: area ratio of high-labeled peptides, non-labeled / labeled;

[0049] M: mass of FIB sample solution in the replicate, mg;

[0050]

[0051] Where C FIB : concentration of FIB in solution; C i : Concentrations of VR, DK, and YR in the solution; M FIB : relative molecular mass of FIB; n: number of corresponding VR, DK, YR fragments in the FIB sequence; M i : Relative molecular weights of VR, DK, and YR.

[0052] Compared with the prior art, the outstanding effects of the present invention are:

[0053] (1) The present invention combines qualitative and quantitative methods to improve the accuracy of the measurement results. A method combining time-of-flight mass spectrometry, high-resolution mass spectrometry, and triple quadrupole tandem mass spectrometry is used to combine qualitative and quantitative methods. Time-of-flight mass spectrometry and high-resolution mass spectrometry select characteristic peptides for quantification by triple quadrupole tandem mass spectrometry. This makes the specific peptides used for quantification by triple quadrupole liquid chromatography-mass spectrometry less susceptible to interference components, thereby improving the accuracy of the quantitative results.

[0054] (2) The method of the present invention determines the value of FIB by selecting the average value of the isotope dilution mass spectrometry results of three characteristic peptide segments, and selecting isotope-labeled amino acids for determination of the three characteristic peptide segments, thereby improving the accuracy of the determination results.

[0055] (3) The present invention uses peptide isotope dilution mass spectrometry to determine the mass concentration of FIB, which is used as the standard value of the reference material. Isotope dilution mass spectrometry is an internationally recognized potential benchmark measurement method. GBW09236 L-valine, GBW09238 L-isoleucine, GBW09239 L-arginine, and GBW09237 L-leucine purity national first-level standard materials are used as reference standards for traceability. Verified / calibrated weighing instruments are used to ensure that the value of the reference material is traceable to the SI basic units of kilogram (kg) and mole (mol).

[0056] The fibrinogen quantification method based on characteristic peptide isotope dilution mass spectrometry of the present invention will be further described below with reference to the accompanying drawings and specific examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is the SDS-page electrophoresis diagram;

[0058] Figure 2 is the liquid chromatogram of FIB;

[0059] Figure 3 This is the TIC diagram of the FIB enzymatic peptide sample;

[0060] Figure 4 Optimization diagram for different enzymatic hydrolysis times;

[0061] Figure 5 Optimization diagram for enzyme amounts at different ratios;

[0062] Figure 6 This is the MRM diagram of the FIB enzymatic peptide. DETAILED DESCRIPTION

[0063] The reagents and instruments used in the following examples include:

[0064] Reagents:

[0065] GBW09237 L-leucine purity standard material: purity 99.7%, U=0.4% (k=2), China National Institute of Metrology.

[0066] GBW09236 L-Valine purity standard material: purity 99.4%, U=0.6% (k=2), China National Institute of Metrology.

[0067] GBW09238 L-isoleucine purity standard material: purity 99.7%, U=0.3% (k=2), China National Institute of Metrology.

[0068] GBW09239 L-arginine purity standard material: purity 99.7%, U=0.5% (k=2), China National Institute of Metrology.

[0069] Isotope-labeled amino acids 13C6-leucine, 13C5-valine, 13C6-isoleucine, and 13C6-arginine were obtained from Cambridge Isotope Laboratory, USA, with a purity of >98%.

[0070] Acetonitrile, Merck, Germany, chromatography grade;

[0071] VR, DK, YR, L-VR, L-DK, and L-YR are from Anhui Guoping Pharmaceutical Co., Ltd.;

[0072] Formic acid, Thermo Fisher Scientific, USA, chromatographic grade;

[0073] Trypsin, Promega, USA;

[0074] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Thermo Fisher Scientific, USA;

[0075] Chloroacetamide (CAA) was purchased from Sigma-Aldrich, USA;

[0076] Sodium deoxycholate (SDC) Shanghai MacLean Biochemical Technology Co., Ltd.;

[0077] Ammonium bicarbonate (NH4HCO3) Sinopharm Chemical Reagent Co., Ltd.

[0078] instrument:

[0079] High performance liquid chromatography triple quadrupole mass spectrometer, AB SCIEX 5500;

[0080] Electronic balance: Sartorius;

[0081] Ultra-high performance liquid chromatograph, Waters BIO H-CLASS.

[0082] Example 1

[0083] Fibrinogen was quantified based on peptide isotope dilution mass spectrometry. The specific steps are as follows:

[0084] 1. Selection of characteristic peptides. Samples were digested with trypsin and analyzed by HPLC triple quadrupole mass spectrometry. The experimental steps are as follows:

[0085] Step (1) Accurately weigh 20 mg of protein solution using an analytical balance, add 180 μL of a mixed solution of 0.5% SDC and 100 mM NH 4 HCO 3 , and then add a mixed solution of 100 mM TCEP and 250 mM CAA, and react at room temperature in the dark for 1 hour.

[0086] Step (2) Add 20 μL of 0.1 mg / mL trypsin solution, mix well, and incubate at 37° C. with shaking for 16 h.

[0087] The product of step (3) was added with 24 μL of 10% formic acid to terminate the enzyme digestion, centrifuged at 14,000 rpm for 20 min, and the supernatant was collected.

[0088] Step (4) Add the labeled peptide at a mass ratio of 1:1, mix well, and centrifuge at 14,000 rpm for 20 min. Take the supernatant and enter the triple quadrupole mass spectrometer to detect the enzyme cleavage product.

[0089] 2. Synthesis of characteristic peptides: The characteristic peptides VR, DK, YR and three isotope-labeled peptides were synthesized. The sequences of the three isotope-labeled peptides were VTSGSTTTTR (13C6, 15N4), DNENVVNEYSSELEK (13C6, 15N2), and YEASILTHDSSIR (13C6, 15N4).

[0090] 3. Accurate quantification of the purity of the characteristic peptides: Using valine, isoleucine, arginine, and leucine standard substances, and isotope-labeled 13C5-valine, 13C6-isoleucine, 13C6-arginine, and 13C6-leucine as internal standards, the three synthesized characteristic peptides were quantified by amino acid isotope dilution mass spectrometry;

[0091] Among them, peptide VR was quantified by isotope dilution mass spectrometry of valine and arginine, DK was quantified by valine and leucine, and YR was quantified by isoleucine and leucine.

[0092] The operation steps are as follows: accurately weigh the prepared VR, DK, and YR solutions respectively and place them in ampoules, record the weighed masses, add equal masses of valine and arginine labeling standard solutions to the VR solution, add equal masses of valine and leucine labeling standard solutions to the DK solution, and add equal masses of isoleucine and leucine labeling standard solutions to the YR solution, concentrate by centrifugation to dryness, then add 800 μL of 6 mol / L hydrochloric acid solution and mix well, deoxygenate and seal with nitrogen, hydrolyze in a 110°C oven for 36 h, take out and blow dry with nitrogen, add 0.1% formic acid-water solution to re-dissolve, filter through a 0.22 μm filter membrane, and wait for detection.

[0093] The content of the synthesized characteristic peptide was calculated using the following formula:

[0094]

[0095] Among them, C AA : concentration of amino acids in solution;

[0096] P: purity of amino acid standards;

[0097] m 标 : The mass of the labeled amino acid added to the peptide, mg;

[0098] R 样 : Amino acid chromatographic peak area ratio in the sample, non-labeled / labeled;

[0099] I1: actual mass ratio of low-labeled amino acids, non-labeled / labeled;

[0100] I2: actual mass ratio of high-standard amino acids, non-labeled / labeled;

[0101] R1: low-label amino acid peak area ratio, non-labeled / labeled;

[0102] R2: peak area ratio of high-labeled amino acids, non-labeled / labeled;

[0103] M: mass of VR, DK, and YR sample solutions in parallel samples, mg;

[0104]

[0105] Where Ci represents the characteristic peptide concentration calculated for each amino acid, CAA represents the concentration of the measured amino acid, Mi represents the relative molecular mass of the characteristic peptide, MAA represents the relative molecular mass of each amino acid, and n represents the number of corresponding amino acids in VR, DK, and YR.

[0106] Liquid phase conditions: Chromatographic column: ACQUITY UPLC Peptide BEH C18, 2.1 mm × 150 mm, 1.7 μm, column temperature 30 ° C, mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile, elution gradient: 0-2 min, 2% B; 2-7 min, 2%-7% B; 7-10 min, 7% B; 10-14 min, 7%-35% B; 14-17 min, 35%-2% B; 17-20 min, 2% B, flow rate 0.2 mL / min, injection volume 5 μL.

[0107] Table 1 Amino acid isotope dilution mass spectrometry conditions

[0108]

[0109]

[0110] Table 2 Peptide purity

[0111] peptides VR DK YR concentration(%) 78.39 77.05 75.82

[0112] 4. The fibrinogen standard was enzymatically digested with trypsin, and the enzymatic digestion conditions were optimized.

[0113] Accurately weigh the internal standard peptide mixed solution. The mass of each peptide in the mixed solution is equivalent to the mass of each peptide after the sample is enzymatically hydrolyzed. Mix the FIB standard solution and the internal standard peptide mixed solution in a mass ratio of 1:1, and optimize at different enzymatic hydrolysis times and different enzyme usage ratios. The enzymatic efficiency is based on the peak area ratio of the enzymatic hydrolyzed peptide to the internal standard peptide. The optimization of different enzymatic hydrolysis times and different enzyme usages is shown in the figure. The final enzymatic hydrolysis time was 16h, and the ratio of sample to trypsin was 10:1. The optimization diagram of different enzymatic hydrolysis times is shown in the figure below. Figure 4 As shown in the figure, the optimization diagram of enzyme amount at different ratios is as follows Figure 5 shown.

[0114] 5. Use peptide isotope dilution mass spectrometry to accurately quantify the three characteristic peptides in the enzymatic hydrolysate. The operation steps are as follows:

[0115] Accurately weigh 20 mg of the diluted protein solution using an analytical balance. Add 180 μL of a mixture of 0.5% SDC and 100 mM NH₄HCO₃, followed by a mixture of 100 mM TCEP and 250 mM CAA. Incubate at room temperature in the dark for 1 hour in the dark. Then, add 20 μL of a 0.1 mg / mL trypsin solution, mix thoroughly, and incubate at 37°C with shaking for 16 hours. Terminate the digestion with 24 μL of 10% formic acid, centrifuge at 14,000 rpm for 20 minutes, remove the supernatant, add the labeled peptide at a 1:1 ratio, mix thoroughly, centrifuge at 14,000 rpm for 20 minutes, and collect the supernatant for detection of the digestion products using a triple quadrupole mass spectrometer.

[0116] 6. Optimization of liquid chromatography and mass spectrometry conditions for three characteristic peptides

[0117] The liquid phase conditions were optimized: mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile. The gradient elution conditions are shown in Table 3.

[0118] Table 3 Mobile phase gradient for peptide isotope dilution mass spectrometry

[0119] Time(min) Flow (mL / min) A(％) B(％) 0 0.2 95 5 15 0.2 50 50 22 0.2 10 90 25 0.2 95 5 30 0.2 95 5

[0120] The mass spectrometry conditions were optimized to determine the parent ions and daughter ions of characteristic peptides and isotope-labeled peptides, and the mass spectrometry parameters were optimized, as shown in Table 4.

[0121] Table 4 Mobile phase mass spectrometry conditions for peptide isotope dilution mass spectrometry

[0122] Q1 Q3 ID DP(V) CE(V) 1010.6 966.5 VR 120 62 885.0 572.2 DK 120 35 746.6 928.6 YR 120 39 1020.5 976.5 L-VR 120 52 889.0 572.1 L-DK 120 36 751.2 938.7 L-YR 120 41

[0123] FIB enzymatic peptide MRM diagram, such as Figure 6 shown.

[0124] 7. Calculate the contents of the three characteristic peptides in the enzymatic hydrolysate. Calculate the concentration of FIB based on the content of the characteristic peptides as the fibrinogen standard. The FIB content is the average of the results of the three characteristic peptides.

[0125] 1) Calculate the mass of the three characteristic peptides after enzyme digestion based on their purity, and prepare the corresponding concentrations of the characteristic peptides and isotope-labeled peptides;

[0126] 2) The three characteristic peptide isotope internal standard solutions were prepared into a mixed solution with a mass ratio of 1:1 to the peptide in the sample solution; the ratios of the peptide standard solution to the isotope internal standard were 0.9:1, 1:1, and 1.1:1;

[0127] 3) Weigh the volume of FIB protein solution to be tested, calculate the mass of the peptide in the FIB sample solution, and add a 1:1 mass ratio of the isotope internal standard mixed solution. The concentration of the characteristic peptide in the FIB to be tested should be in the middle of the determined linear range.

[0128] 4) Calculate the concentration of FIB using the following formula:

[0129]

[0130] Among them, C i : Concentrations of VR, DK, and YR in the solution;

[0131] P: purity of characteristic peptides VR, DK, and YR;

[0132] m 标 : The mass of the labeled peptide added to the characteristic peptide, mg;

[0133] R 样 : chromatographic peak area ratio of characteristic peptides in the sample, non-labeled / labeled;

[0134] I1: actual mass ratio of low-label peptide, non-labeled / labeled;

[0135] I2: actual mass ratio of high-labeled peptides, non-labeled / labeled;

[0136] R1: low-label peptide peak area ratio, non-labeled / labeled;

[0137] R2: area ratio of high-labeled peptides, non-labeled / labeled;

[0138] M: mass of FIB sample solution in the replicate, mg;

[0139]

[0140] Where C FIB : concentration of FIB in solution; C i : Concentrations of VR, DK, and YR in the solution; M FIB : relative molecular mass of FIB; n: number of corresponding VR, DK, YR fragments in the FIB sequence; M i : The relative molecular masses of VR, DK, and YR. The determination result of fibrinogen is the average value of the determination values ​​of the three characteristic peptides VR, DK, and YR.

[0141] Table 5 Mobile phase mass spectrometry conditions for peptide isotope dilution mass spectrometry

[0142]

[0143] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A fibrinogen quantification method based on characteristic peptide isotope dilution mass spectrometry, characterized in that: The following steps are involved: Step (1) Selection of characteristic peptides: VR, DK, and YR are selected as characteristic peptides, and the sequences of VR, DK, and YR are VTSGSTTTTR, DNENVVNEYSSELEK, and YEASILTHDSSIR, respectively; Step (2) Synthesis of characteristic peptides: Synthesize characteristic peptides VR, DK, YR and three isotope-labeled peptides, the sequences of the three isotope-labeled peptides are VTSGSTTTTR (13C6, 15N4), DNENVVNEYSSELEK (13C6, 15N2), and YEASILTHDSSIR (13C6, 15N4). Step (3) Accurate quantification of the purity of the characteristic peptide segment: the synthesized peptide segment is hydrolyzed into amino acids, isotope-labeled amino acids are quantitatively added, and the amino acid isotope dilution mass spectrometry method is established using amino acid standard substances as standards to determine the content of stable amino acids in the hydrolyzate, and the purity of the characteristic peptide segment is calculated accordingly; Step (4) FIB is enzymatically digested with trypsin to obtain fibrinogen; Step (5) using peptide isotope dilution mass spectrometry to accurately quantify the three characteristic peptides in the enzymatic hydrolysate; Step (6) Optimization of liquid phase conditions and mass spectrometry conditions for three characteristic peptides: determining the parent ions and daughter ions of the characteristic peptides and isotope-labeled peptides, and optimizing the mass spectrometry parameters; Step (7) calculates the contents of the three characteristic peptides in the enzymatic hydrolysate respectively, and calculates the concentration of FIB based on the contents of the characteristic peptides as the fixed value result of the protein.

2. The method for quantifying fibrinogen based on characteristic peptide isotope dilution mass spectrometry according to claim 1, characterized in that: VR, DK, and YR were selected as characteristic peptides.

3. The method for quantifying fibrinogen based on characteristic peptide isotope dilution mass spectrometry according to claim 1, characterized in that: In the step (3), the peptide VR is quantified by isotope dilution mass spectrometry of valine and arginine, DK is quantified by valine and leucine, and YR is quantified by isoleucine and leucine.

4. The method for quantifying fibrinogen based on characteristic peptide isotope dilution mass spectrometry according to claim 1, characterized in that: In the step (4), the enzymatic hydrolysis time is 16 h, and the ratio of sample to trypsin is 10:

1.

5. The method for quantifying fibrinogen based on characteristic peptide isotope dilution mass spectrometry according to claim 1, characterized in that: In the step (5), the content of the synthesized characteristic peptide segment is calculated using the following formula: Among them, C AA : concentration of amino acids in solution; P: purity of amino acid standards; m 标 : The mass of the labeled amino acid added to the peptide, mg; R 样 : Amino acid chromatographic peak area ratio in the sample, non-labeled / labeled; I1: actual mass ratio of low-labeled amino acids, non-labeled / labeled; I2: actual mass ratio of high-standard amino acids, non-labeled / labeled; R1: low-label amino acid peak area ratio, non-labeled / labeled; R2: peak area ratio of high-labeled amino acids, non-labeled / labeled; M: mass of VR, DK, and YR sample solutions in parallel samples, mg; Where C i represents the characteristic peptide concentration calculated for each amino acid, C AA Represents the concentration of the measured amino acid, M i Represents the relative molecular mass of the characteristic peptide segment, M AA represents the relative molecular mass of each amino acid, and n represents the number of corresponding amino acids in VR, DK, and YR.

6. The method for quantifying fibrinogen based on characteristic peptide isotope dilution mass spectrometry according to claim 1, characterized in that: In step (6), the chromatographic column used is ACQUITY UPLC Peptide BEH C18, 2.1 mm × 150 mm, 1.7 μm; Mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, the flow rate was 0.2 ml / min, and the injection volume was 5 μL; Flow gradient: 0-15 min, 5%-50% B; 15-22 min, 50%-90% B; 22-25 min, 90%-5% B; 25-30 min, 5% B; VR 1010.6 / 966.5, DK 885.0 / 572.2, YR 746.6 / 928.6, 13C-VR 1020.5 / 976.5, 13C-DK 889.0 / 572.1, 13C-YR 751.2 / 938.7; and the DP and CE values ​​were optimized.

7. The method for quantifying fibrinogen based on characteristic peptide isotope dilution mass spectrometry according to claim 1, characterized in that: The step (7) is specifically as follows: 1) Calculate the mass of the three characteristic peptides after enzyme digestion based on their purity, and prepare the corresponding concentrations of the characteristic peptides and isotope-labeled peptides; 2) The three characteristic peptide isotope internal standard solutions were prepared into a mixed solution with a mass ratio of 1:1 to the peptide in the sample solution; the ratios of the peptide standard solution to the isotope internal standard were 0.9:1, 1:1, and 1.1:1; 3) Weigh the volume of FIB protein solution to be tested, calculate the mass of the peptide in the FIB sample solution, and add a 1:1 mass ratio of the isotope internal standard mixed solution. The concentration of the characteristic peptide in the FIB to be tested should be in the middle of the determined linear range. 4) Calculate the concentration of FIB using the following formula: Among them, C i : Concentrations of VR, DK, and YR in the solution; P: purity of characteristic peptide; m 标 : The mass of the labeled peptide added to the characteristic peptide, mg; R 样 : chromatographic peak area ratio of characteristic peptides in the sample, non-labeled / labeled; I1: actual mass ratio of low-label peptide, non-labeled / labeled; I2: actual mass ratio of high-labeled peptides, non-labeled / labeled; R1: low-label peptide peak area ratio, non-labeled / labeled; R2: area ratio of high-labeled peptides, non-labeled / labeled; M: mass of FIB sample solution in the replicate, mg; Where C FIB : concentration of FIB in solution; C i : Concentrations of VR, DK, and YR in the solution; M FIB : relative molecular mass of FIB; n: number of corresponding VR, DK, YR fragments in the FIB sequence; M i : Relative molecular weights of VR, DK, and YR.

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