Characteristic peptide fragment group for SAA protein valuing and application thereof

Abstract

The invention relates to a characteristic peptide fragment group for SAA protein valuing and application of the characteristic peptide fragment group. The characteristic peptide fragment group comprises a sequence 1, a sequence 1 of an I site connected isotope, a sequence 2 and a sequence 2 of an F site connected isotope, the amino acid sequence of the sequence 1 comprises a sequence as shown in SEQ ID NO.1, and the amino acid sequence of the sequence 2 comprises a sequence as shown in SEQ ID NO.2. According to the method, the isotope labeled characteristic peptide fragment is introduced as an internal standard, so that the loss in the sample treatment process and the signal fluctuation in the mass spectrometric detection process are effectively corrected, and the influence of a matrix effect on a quantitative result is overcome, so that the quantitative accuracy of the SAA protein is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein detection, and particularly relates to a characteristic peptide segment group for SAA protein quantification and application thereof. BACKGROUND

[0002] Serum amyloid A (SAA) is an important acute phase reactant protein, which plays a key role in various physiological and pathological processes such as inflammation, infection and tumor. Accurate determination of the content of SAA protein is of great significance for early diagnosis of diseases, disease monitoring and evaluation of treatment effect. At present, the methods for quantifying SAA protein mainly include immunological methods and traditional mass spectrometry methods.

[0003] Immunological methods, such as enzyme-linked immunosorbent assay (ELISA) and immunoturbidimetry, are commonly used for quantification of SAA protein in clinical practice. However, immunological methods have some significant limitations. On the one hand, the detection results are easily affected by factors such as antibody specificity, cross-reaction and interfering substances in the sample, resulting in poor accuracy and repeatability of the detection results. The detection results of reagent kits produced by different manufacturers and different batches may have large differences, making it difficult to standardize and compare the detection results between different laboratories. On the other hand, immunological methods can only determine the total amount of SAA protein, and cannot accurately distinguish and quantify different subtypes of SAA protein, which brings certain difficulties to the in-depth study of the specific mechanism of SAA protein in different diseases and the realization of more accurate disease diagnosis and treatment.

[0004] Traditional mass spectrometry methods, such as liquid chromatography-mass spectrometry (LC-MS) technology, have high accuracy and specificity in protein quantification analysis. However, traditional mass spectrometry methods also have some technical problems. First, the traditional mass spectrometry method is easily affected by the matrix effect in the quantification process. Other components in the sample may interfere with the ionization efficiency of the target peptide segment, causing fluctuations in the detection signal, thereby affecting the accuracy of quantification. Second, the traditional mass spectrometry method has high requirements for sample pretreatment, which requires complex extraction and purification steps to remove impurities and interfering substances in the sample, which not only increases the complexity and time cost of experimental operation, but also may introduce errors in the sample processing process, affecting the reliability of the final detection results.

[0005] Therefore, how to construct a method for accurately quantifying SAA protein has become a problem to be solved at present. SUMMARY

[0006] To solve the above technical problems, the application provides a characteristic peptide segment group for SAA protein quantification and application thereof, which introduces an isotopically labeled characteristic peptide segment as an internal standard, effectively corrects loss in sample processing and signal fluctuation in mass spectrometric detection, overcomes the influence of matrix effect on quantitative results, and significantly improves the accuracy of SAA protein quantification.

[0007] To achieve the above purpose, the application adopts the following technical solutions.

[0008] In a first aspect, the application provides a characteristic peptide segment group for SAA protein quantification, which comprises sequence 1, sequence 1 connected with an isotope at an I (isoleucine) site, sequence 2, and sequence 2 connected with an isotope at an F (phenylalanine) site; the amino acid sequence of sequence 1 comprises the sequence shown in SEQ ID NO. 1, and the amino acid sequence of sequence 2 comprises the sequence shown in SEQ ID NO. 2.

[0009] SEQ ID NO. 1: EANYIGSDK.

[0010] SEQ ID NO. 2: SFFSFLGEAFDGAR.

[0011] In the application, EANYIGSDK and SFFSFLGEAFDGAR are selected as characteristic peptide segments, compared with other peptide segments, SAA protein can stably generate the two peptide segments of sequence 1 and sequence 2 after being digested by trypsin, and the signal intensity is strong in mass spectrometric detection, which can improve the sensitivity of detection.

[0012] Preferably, the isotope connected at the I site comprises 13 C and / or 15 N.

[0013] Preferably, the isotope connected at the F site comprises 13 C and / or 15 N.

[0014] In a second aspect, the application provides application of the characteristic peptide segment group for SAA protein quantification according to the first aspect in quantifying SAA protein.

[0015] In a third aspect, the application provides a method for quantifying SAA protein based on characteristic peptide segment isotope dilution mass spectrometry, which comprises mixing a sample solution of SAA protein to be detected and the isotope-connected peptide segment in the characteristic peptide segment group according to the first aspect, and then performing mass spectrometric detection.

[0016] Preferably, the method comprises: using mass spectrometric detection on the sample solution, the high-concentration solution, and the low-concentration solution to calculate the content of sequence 1 and sequence 2 in the SAA protein sample.

[0017] The preparation method of the sample solution is mixing the SAA protein sample solution to be detected, sequence 1 connected with the I-site isotope and sequence 2 connected with the F-site isotope, then adding trypsin and ammonium bicarbonate to obtain the sample solution.

[0018] The preparation method of the high standard solution is mixing sequence 1, sequence 1 connected with the I-site isotope, sequence 2 and sequence 2 connected with the F-site isotope.

[0019] The preparation method of the low standard solution is mixing sequence 1, sequence 1 connected with the I-site isotope, sequence 2 and sequence 2 connected with the F-site isotope.

[0020] Preferably, the content calculation formula of sequence 1 and sequence 2 in the SAA protein sample is as follows:

[0021] ;

[0022] Wherein, is the content of sequence 1 or sequence 2 in the SAA protein sample; is the mass of sequence 1 connected with the isotope or sequence 2 connected with the isotope in the sample solution; is the peak area ratio of sequence 1 and sequence 1 connected with the isotope or sequence 2 and sequence 2 connected with the isotope in the sample solution; is the mass ratio of sequence 1 and sequence 1 connected with the isotope or sequence 2 and sequence 2 connected with the isotope in the high standard solution; is the mass ratio of sequence 1 and sequence 1 connected with the isotope or sequence 2 and sequence 2 connected with the isotope in the low standard solution; is the peak area ratio of sequence 1 and sequence 1 connected with the isotope or sequence 2 and sequence 2 connected with the isotope in the high standard solution; is the peak area ratio of sequence 1 and sequence 1 connected with the isotope or sequence 2 and sequence 2 connected with the isotope in the low standard solution; is the mass of the SAA protein sample; is the concentration of sequence 1 or sequence 2.

[0023] In the present application, the content detection method is used to detect the content of sequence 1 or sequence 2 in the SAA protein sample. ;

[0024] Wherein, c is the content of sequence 1 or sequence 2 in the SAA protein sample, is the molecular weight of the SAA protein, is the molecular weight of sequence 1 or sequence 2, is the content of the SAA protein.

[0025] Preferably, the content of SAA protein in the sample solution is calculated as the average of the content of SAA protein calculated using sequence 1 and sequence 2, respectively.

[0026] In the present application, the concentration of SAA protein in the solution is calculated according to the molecular weight of SAA protein and characteristic peptide segment as the valuation result of the standard substance. Specifically, the concentrations of sequence 1 and sequence 2 in SAA calculated by the above formula are c1 and c2, respectively, and the SAA content is derived by multiplying c1 by the molecular weight of SAA and dividing by the molecular weight of sequence 1, and the SAA content is derived by multiplying c2 by the molecular weight of SAA and dividing by the molecular weight of sequence 2, and the average content of SAA is calculated by averaging the two contents.

[0027] Preferably, the mass ratio of sequence 1, sequence 1 connected to isotope at I site, sequence 2, and sequence 2 connected to isotope at F site in the high standard solution is 1:(0.8-1.5):(1-5):(0.8-1.5). The (0.8-1.5), for example, can be 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or 1.5, etc. The (1-5), for example, can be 1, 2, 3, 4, or 5, etc.

[0028] Preferably, the mass ratio of sequence 1, sequence 1 connected to isotope at I site, sequence 2, and sequence 2 connected to isotope at F site in the low standard solution is 1:(1-2):(1-5):(1-2). The (1-2), for example, can be 1, 1.2, 1.4, 1.6, 1.8, or 2, etc. The (1-5), for example, can be 1, 2, 3, 4, or 5, etc.

[0029] Preferably, the mass ratio of SAA protein sample, sequence 1 connected to isotope at I site, and sequence 2 connected to isotope at F site in the sample solution is 1:(0.5-3):(0.5-3). The (0.5-3), for example, can be 0.5, 1, 1.5, 2, 2.5, or 3, etc.

[0030] Preferably, the time for enzyme digestion is 16-48 h, and the temperature is 35-40℃. The 16-48 h, for example, can be 16 h, 20 h, 25 h, 30 h, 35 h, or 48 h, etc. The 35-40℃, for example, can be 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃, etc.

[0031] Preferably, the mass ratio of SAA protein to trypsin is (5-25):1. The (5-25), for example, can be 5, 10, 15, 20, or 25, etc.

[0032] Preferably, the concentration of the ammonium bicarbonate is 1-10 mM. The 1-10 mM, for example, can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM, etc.

[0033] Compared with the prior art, the present application has at least the following beneficial effects:

[0034] 1. In the present application, the SAA protein can be stably generated after trypsin digestion to generate two characteristic peptide segments of sequence 1 and sequence 2 in the characteristic peptide segment group, and the signal intensity is strong in mass spectrometry detection, which can improve the sensitivity of detection.

[0035] 2. The method of the present application introduces isotopically labeled characteristic peptide segments as internal standards to effectively correct the loss during sample processing and signal fluctuations during mass spectrometry detection, overcome the influence of matrix effect on quantitative results, and significantly improve the accuracy of SAA protein quantification, providing more reliable basis for precise diagnosis and treatment of diseases.

[0036] 3. The present application establishes a standardized experimental process and quality control system to ensure the consistency and comparability of detection results between different batches of experiments and different laboratories, reduce experimental errors, improve the repeatability of quantitative results, and make the SAA protein quantitative results can be recognized and applied in a wider range.

[0037] 4. The present application optimizes the sample pretreatment method, reduces unnecessary operation steps, reduces the complexity and time cost of experimental operation, and improves the efficiency and stability of sample processing, making the method more suitable for large-scale clinical detection and scientific research application. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a mass spectrometry peptide map generated after trypsin digestion of SAA protein.

[0039] Figure 2 is a liquid chromatogram of polypeptide EK.

[0040] Figure 3 is a mass spectrometry peptide map of polypeptide EK.

[0041] Figure 4 is a liquid chromatogram of polypeptide EK-IS.

[0042] Figure 5 is a mass spectrometry peptide map of polypeptide EK-IS.

[0043] Figure 6 is a liquid chromatogram of polypeptide SR.

[0044] Figure 7 is a mass spectrometry peptide map of polypeptide SR.

[0045] Figure 8 This is the liquid chromatogram of the polypeptide SR-IS.

[0046] Figure 9 This is the SR-IS mass spectrum of the polypeptide.

[0047] Figure 10 This is a linear graph of the characteristic peptide EK.

[0048] Figure 11 This is a linear plot of the characteristic peptide SR.

[0049] Figure 12 This is an optimized diagram of the cone-hole voltage for characteristic peptide segments.

[0050] Figure 13 This is a diagram showing the optimized collision energy of characteristic peptide segments.

[0051] Figure 14 This is a linear graph of the characteristic peptide EK.

[0052] Figure 15 This is a linear plot of the characteristic peptide SR. Detailed Implementation

[0053] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0054] Example 1

[0055] This embodiment involves screening for characteristic peptides.

[0056] First, the SAA protein sequence was input into the Expasy database. Using the PeptideMass tool, trypsin was selected to generate the theoretical polypeptide sequence and corresponding molecular weight of the SAA protein after trypsin digestion, as shown in Table 1. Then, the SAA protein sample was digested with trypsin and analyzed by time-of-flight mass spectrometry to generate the actual polypeptide sequence and corresponding molecular weight, as shown in Table 1. Figure 1 As shown. Matching Table 1 and Figure 1 Based on the data, two characteristic peptides, EANYIGSDK and SFFSFLGEAFDGAR, were selected. Subsequent triple quadrupole mass spectrometry analysis showed that these two peptides exhibited good precursor / daughter ion signals and their migration times during liquid-phase separation were consistent with those of the isotope-labeled internal standard.

[0057] Table 1

[0058]

[0059] Example 2

[0060] This embodiment involves the synthesis of characteristic peptides.

[0061] Two characteristic peptides, EANYIGSDK (named EK) and SFFSFLGEAFDGAR (named SR), were synthesized, along with two isotope-labeled peptides, EANYI*GSDK, with isotopes linked at the I site. 13 C (named EK-IS) and SFFSF*LGEAFDGAR, with isotopes linked at the F site. 13 C (named SR-IS).

[0062] The purity of characteristic peptides was determined by high performance liquid chromatography (HPLC), and the molecular weight of characteristic peptides was determined by time-of-flight mass spectrometry (TOF-MS). Specific results are as follows: Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Figure 9 As shown in Table 2, the purity and molecular weight of the peptides are consistent with the theoretical molecular weight.

[0063] The liquid chromatography conditions were as follows: Kinetex C18 column (100 mm × 2.1 mm), mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 100% acetonitrile, column temperature 30℃, sample chamber temperature 10℃, and flow rate 0.2 mL / min.

[0064] The elution gradient is as follows (the total concentration of mobile phase A and mobile phase B is 100%):

[0065] 0 min, mobile phase A is 95%;

[0066] Within 0-5 minutes, the mobile phase A changed from 95% to 75% at a constant rate.

[0067] Over 5-25 minutes, the mobile phase A decreased from 75% to 20% at a constant rate.

[0068] Over 25-25.1 min, the mobile phase A changed from 20% to 95% at a constant rate.

[0069] 25.1-30 min, mobile phase A is 95%;

[0070] The mass spectrometry conditions were as follows: positive ion detection; multiple reaction monitoring (MRM) scanning mode; spray voltage: 1.0 kV; ion source temperature: 150℃; desolvation gas temperature: 500℃; desolvation gas flow rate: 900 L / h; cone gas path flow rate: 150 L / h.

[0071] Table 2

[0072]

[0073] The purity of the above-mentioned characteristic peptides is greater than 90%, and they can be used for subsequent experiments.

[0074] Example 3

[0075] This embodiment explores the linear range of characteristic peptide segments in liquid chromatography-mass spectrometry.

[0076] The synthesized peptides were hydrolyzed into amino acids, and isotopically labeled amino acids were added quantitatively. An amino acid hydrolysis isotope dilution mass spectrometry method was established using national amino acid standard materials to determine the content of stable amino acids in the hydrolysate and calculate the concentration of characteristic peptides. Isoleucine (I) was used for quantification of the characteristic peptide EK, and phenylalanine (F) was used for quantification of the characteristic peptide SR.

[0077] The concentrations of the characteristic peptide stock solution were determined, and two characteristic peptide solutions were prepared into peptide solutions of 9.6 μg / g and 4.5 μg / g, respectively. These solutions were then added to the synthesized labeled peptide solution and the aforementioned standard solution at a mass ratio of 0.8–1.2 and thoroughly mixed. Based on the peak area ratio of the standard peptide to the labeled peptide, the linearity within the range of 0.8–1.2 was calculated, as detailed below. Figure 10 and Figure 11 As shown in the figure. The results indicate that the two peptides exhibit good linearity (r > 0.999) in a mass ratio between 0.8 and 1.2, making this range suitable for protein determination.

[0078] Example 4

[0079] This embodiment optimizes mass spectrometry parameters and enzyme digestion parameters.

[0080] (1) Optimization of mass spectrometry parameters

[0081] EK, EK-IS, SR, and SR-IS were prepared into a mixed solution and then detected by mass spectrometry. The concentrations of EK, EK-IS, SR, and SR-IS in the solution were 0.000375 μg / g, 0.00045 μg / g, 0.00058 μg / g, and 0.0006 μg / g, respectively. Mass spectrometry parameters were optimized as follows: positive ion detection; multiple reaction monitoring (MRM); spray voltage: 1.0 kV; ion source temperature: 150℃; desolvation gas temperature: 500℃; desolvation gas flow rate: 900 L / h; orifice gas flow rate: 150 L / h. The orifice voltage and collision energy parameters were also optimized. Specific results are shown below. Figure 12 and Figure 13As shown, the cone voltage and collision energy at which the EK and EK-IS mass spectra reached their highest intensities were 50 V and 14 V, respectively; the cone voltage and collision energy at which the SR mass spectra reached their highest intensities were 20 V and 20 V, respectively; and the cone voltage and collision energy at which the SR-IS mass spectra reached their highest intensities were 40 V and 20 V, respectively. The cone voltage and collision energy at which the mass spectra reached their highest intensities were selected as the optimal experimental parameters. Therefore, the ion pairs and optimal mass spectra parameters for the characteristic peptides are shown in Table 3.

[0082] Table 3

[0083]

[0084] (2) Optimization of enzyme digestion parameters

[0085] 50 mg of SAA protein, 50 mg of EK-IS, and 50 mg of SR-IS were mixed, and 10 mg of trypsin was added. Then, ammonium bicarbonate was added to a final concentration of 5 mM. The mixture was digested at 37°C for 8 h, 16 h, 24 h, 40 h, and 48 h, respectively. Mass spectrometry analysis was then performed using optimized parameters. Specific results are shown below. Figure 14 As shown.

[0086] 50 mg of SAA protein, 50 mg of EK-IS, and 50 mg of SR-IS were mixed, and then ammonium bicarbonate was added to a final concentration of 5 mM. 0.5 mg, 1 mg, 2 mg, 5 mg, and 10 mg of trypsin were added, and the mixture was digested at 37°C for 16 h. Mass spectrometry analysis was then performed using optimized parameters. Specific results are shown below. Figure 15 As shown.

[0087] The results above indicate that enzyme digestion is most effective when the digestion time is between 16 h and 48 h, and the optimal ratio of trypsin to SAA protein is 1:(5-25).

[0088] Example 5

[0089] This embodiment performs SAA protein level determination.

[0090] Sample solution: Mix 50 mg SAA protein, 50 mg EK-IS and 50 mg SR-IS, add 10 mg trypsin, add ammonium bicarbonate to a final concentration of 5 mM, and digest at 37℃ for 16 h.

[0091] High-standard solution: EK, SR, EK-IS and SR-IS were mixed. The concentration of EK in the mixed solution was 0.000422 μg / g, the concentration of EK-IS was 0.0004 μg / g, the concentration of SR was 0.00064 μg / g and the concentration of SR-IS was 0.0006 μg / g, that is, the ratio of EK:EK-IS:SR:SR-I was 1:0.95:1.52:1.42.

[0092] Low-standard solution: EK, SR, EK-IS and SR-IS were mixed. The concentration of EK in the mixed solution was 0.000346 μg / g, the concentration of EK-IS was 0.0004 μg / g, the concentration of SR was 0.00053 μg / g and the concentration of SR-IS was 0.0006 μg / g, that is, the ratio of EK:EK-IS:SR:SR-IS was 1:1.16:1.53:1.74.

[0093] The above solution was analyzed by mass spectrometry using the optimized mass spectrometry parameters, and the concentration was detected using the high performance liquid chromatography parameters of Example 2.

[0094] The detected structure is then substituted into the content calculation formula for calculation:

[0095] ;

[0097] in, The content of sequence 1 or sequence 2 in the SAA protein sample; The mass of either isotope-linked sequence 1 or isotope-linked sequence 2 in the sample solution; It is the peak area ratio of sequence 1 to isotope-linked sequence 1 in the sample solution, or the peak area ratio of sequence 2 to isotope-linked sequence 2. The mass ratio of sequence 1 to isotope-linked sequence 1 in the high-standard solution, or the mass ratio of sequence 2 to isotope-linked sequence 2; This refers to the mass ratio of sequence 1 to isotope-linked sequence 1 in the low-standard solution, or the mass ratio of sequence 2 to isotope-linked sequence 2. It is the peak area ratio of high-standard solution sequence 1 to isotope-linked sequence 1, or the peak area ratio of sequence 2 to isotope-linked sequence 2. It is the peak area ratio of low-standard solution sequence 1 to isotope-linked sequence 1, or the peak area ratio of sequence 2 to isotope-linked sequence 2. For SAA protein sample quality; The concentration of sequence 1 or sequence 2.

[0098] The specific detection results are shown in Tables 4 and 5. Table 4 shows the concentrations of sequence 1 and sequence 2, and Table 5 shows the concentration of SAA protein calculated after substituting into the formula.

[0099] Table 4

[0100]

[0101] Table 5

[0102]

[0103] Example 6

[0104] This embodiment explores the effects of different peptides on efficacy.

[0105] SAA protein was quantified using the GPGGVWAAEAISDAR peptide and the GP*GGVWAAEAISDAR isotope internal standard peptide, with P-site ligation. 13 C. The detected content of GPGGVWAAEAISDAR peptides in SAA, as shown in Tables 6 and 7, was less than one-tenth of the theoretically calculated content. For specific experimental procedures, refer to the EK, EK-IS, and SR, SR-IS methods for determining SAA protein content.

[0106] Table 6

[0107]

[0108] Table 7

[0109]

[0110] In summary, this invention, by introducing isotopically labeled characteristic peptides as internal standards, effectively corrects for losses during sample processing and signal fluctuations during mass spectrometry detection, overcomes the influence of matrix effects on quantitative results, and thus significantly improves the accuracy of SAA protein quantification.

[0111] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A set of characteristic peptides for SAA protein assay, characterized in that, The characteristic peptide group includes sequence 1, sequence 1 with an isotope linked at site I, sequence 2, and sequence 2 with an isotope linked at site F; The amino acid sequence of sequence 1 includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of sequence 2 includes the sequence shown in SEQ ID NO.

2.

2. The characteristic peptide set for SAA protein quantification according to claim 1, characterized in that, The isotopes linked at site I include 13 C and / or 15 N; Preferably, the isotope linked to the F site includes 13 C and / or 15 N.

3. The application of the characteristic peptide group for SAA protein quantification according to claim 1 or 2 in quantifying SAA protein.

4. A method for determining the value of SAA protein based on characteristic peptide isotope dilution mass spectrometry, characterized in that, The method includes mixing the SAA protein sample solution to be tested with the linker isotope peptides in the characteristic peptide group of claim 1 or 2 and then performing mass spectrometry detection.

5. The method for determining the value of SAA protein based on characteristic peptide isotope dilution mass spectrometry according to claim 4, characterized in that, The method includes: calculating the content of sequence 1 and sequence 2 in the SAA protein sample after mass spectrometry detection of the sample solution, high-standard solution and low-standard solution; The sample solution is prepared by adding sequence 1 (linked to isotope at site I) and sequence 2 (linked to isotope at site F) to the sample solution of the SAA protein to be tested, mixing them, and then adding trypsin and ammonium bicarbonate for digestion to obtain the sample solution. The high-standard solution is prepared by mixing sequence 1, sequence 2 with isotope linked at the I site, and sequence 2 with isotope linked at the F site; The method for preparing the low-standard solution is to mix Sequence 1 (linked to isotopes at the I site), Sequence 2 (linked to isotopes at the F site), and Sequence 2 (linked to isotopes at the F site).

6. The method for determining the value of SAA protein based on characteristic peptide isotope dilution mass spectrometry according to claim 5, characterized in that, The formula for calculating the content of sequence 1 and sequence 2 in the SAA protein sample is as follows: ； in, The content of sequence 1 or sequence 2 in the SAA protein sample; The mass of either isotope-linked sequence 1 or isotope-linked sequence 2 in the sample solution; It is the peak area ratio of sequence 1 to isotope-linked sequence 1 in the sample solution, or the peak area ratio of sequence 2 to isotope-linked sequence 2. The mass ratio of sequence 1 to isotope-linked sequence 1 in the high-standard solution, or the mass ratio of sequence 2 to isotope-linked sequence 2; This refers to the mass ratio of sequence 1 to isotope-linked sequence 1 in the low-standard solution, or the mass ratio of sequence 2 to isotope-linked sequence 2. It is the peak area ratio of high-standard solution sequence 1 to isotope-linked sequence 1, or the peak area ratio of sequence 2 to isotope-linked sequence 2. It is the peak area ratio of low-standard solution sequence 1 to isotope-linked sequence 1, or the peak area ratio of sequence 2 to isotope-linked sequence 2. For SAA protein sample quality; The concentration of sequence 1 or sequence 2; Preferably, the formula for determining the value of the SAA protein is as follows: ; Where c represents the content of sequence 1 or sequence 2 in the SAA protein sample. This represents the molecular weight of the SAA protein. The molecular weight is either sequence 1 or sequence 2. This refers to the content of SAA protein.

7. The method for determining the value of SAA protein based on characteristic peptide isotope dilution mass spectrometry according to claim 5 or 6, characterized in that, The mass ratio of Sequence 1, Sequence 2 (linked to isotopes at site I), and Sequence 2 (linked to isotopes at site F) in the high-standard solution is 1:(0.8-1.5):(1-5):(0.8-1.5). Preferably, the mass ratio of Sequence 1, Sequence 2 with I site linked to isotope and Sequence 2 with F site linked to isotope in the low-standard solution is 1:(1-2):(1-5):(1-2).

8. The method for determining the value of SAA protein based on characteristic peptide isotope dilution mass spectrometry according to any one of claims 5-7, characterized in that, The mass ratio of the SAA protein sample, the isotope-linked sequence 1 at site I, and the isotope-linked sequence 2 at site F in the sample solution is 1:(0.5-3):(0.5-3).

9. The method for determining the value of SAA protein based on characteristic peptide isotope dilution mass spectrometry according to any one of claims 5-8, characterized in that, The enzyme digestion time is 16-48 h at 35-40℃.

10. The method for determining the value of SAA protein based on characteristic peptide isotope dilution mass spectrometry according to any one of claims 5-9, characterized in that, The mass ratio of SAA protein to trypsin is (5-25):1.

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