Method for lc-ms / ms quantitative detection of mucinophilic akkermansia muciniphila p9 protein based on characteristic polypeptides

Abstract

The present application relates to a method for detecting P9 protein of mucinophilic Akkermansia based on characteristic polypeptide LC-MS / MS. The method realizes high-precision quantitative detection of P9 protein in AKK sample through sample pretreatment, trypsin digestion, LC-MS / MS detection and standard curve quantification. The present application does not depend on antibodies, has higher specificity and accuracy, and the linear range is 1-1000 ng / mL. The actual measured content of P9 protein is greater than or equal to 3 ng / mg, which is qualified. The present application is suitable for precise quantitative analysis and product quality control of P9 protein of AKK.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a quantitative detection method for Akkermansia p9 protein based on characteristic polypeptides using LC-MS / MS. Background Technology

[0002] The P9 protein is a core functional protein in the metabolic regulation of AKK bacteria, and its content directly reflects the functional activity of AKK bacteria products. Existing methods for P9 protein detection mainly include immunoassay (ELISA), but ELISA relies on the availability of specific antibodies, and batch-to-batch antibody variability may affect the accuracy of the results. Mass spectrometry (LC-MS / MS), as a high-precision quantitative method, does not rely on antibodies and has higher specificity and accuracy. Currently, there is no LC-MS / MS quantitative detection method based on the characteristic peptides of the P9 protein. Summary of the Invention

[0003] ① Problem to be solved: To provide a high-precision quantitative detection method for P9 protein that does not rely on antibodies, so as to overcome the technical defects of existing immunological detection methods, such as large batch-to-batch antibody variability and limited accuracy.

[0004] ② Technical Solution: This invention provides a method for quantitative detection of AKK strain P9 protein by LC-MS / MS based on characteristic peptides, comprising: sample pretreatment (treatment of lysis buffer containing denaturant and reducing agent) → trypsin digestion (obtaining characteristic peptides of P9 protein) → LC-MS / MS detection (MRM mode) → quantitative analysis (stable isotope-labeled internal standard or standard curve). The characteristic peptides are selected from YLNDQVAK (m / z 472.25), FVSEAIGK (m / z 438.24), TAAYILGQK (m / z 501.27), or VVNEALSK (m / z 452.26).

[0005] ③ Beneficial effects: It does not depend on antibodies, avoiding batch-to-batch antibody variability; it has high detection accuracy and specificity, with a linear range of 1-1000 ng / mL; it can be used as a reference or confirmatory method for immunological detection. Detailed Implementation

[0006] Example 1: Screening and identification of characteristic peptides of P9 protein Recombinant P9 protein (theoretical molecular weight approximately 25.3 kDa) was digested with trypsin, and the digestion products were analyzed by LC-MS / MS. By comparing the amino acid sequence of the P9 protein with the mass spectrometry data, the following four characteristic polypeptide sequences were identified: Peptide number sequence, precursor ion m / z, retention time (min), specificity P9-P1 YLNDQVAK 472.25 (2+) 12.3 P9 protein specific P9-P2 FVSEAIGK 438.24 (2+) 14.1 P9 protein specific P9-P3 TAAYILGQK 501.27 (2+) 15.8 P9 protein specific P9-P4 VVNEALSK 452.26 (2+) 11.5 P9 protein specific Example 2: LC-MS / MS detection method (1) Sample pretreatment: Take 100 mg of AKK lyophilized powder, add 1 mL of extraction buffer (8M urea, 50 mM NH4HCO3, 10 mM DTT), and extract with shaking at room temperature for 2 hours. Centrifuge at 12,000 g for 20 minutes at 4℃ and collect the supernatant. Add iodoacetamide to a final concentration of 20 mM and alkylate at room temperature in the dark for 30 minutes. Add trypsin (enzyme:protein = 1:50) and enzymatically digest overnight (16 hours) at 37℃. Desalt the enzymatic digest by C18 solid-phase extraction column, freeze-dry, and then reconstitute in 100 μL of 0.1% formic acid aqueous solution.

[0007] (2) Instrument conditions: The liquid chromatography system was UHPLC, and the chromatographic column was ACQUITY UPLC BEH C18 (2.1×100mm, 1.7μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Gradient elution program: 0-2min 5%B, 2-25min 5-40%B, 25-26min 40-95%B, 26-28min 95%B, 28-28.5min 95-5%B, 28.5-32min 5%B. Flow rate: 0.3 mL / min, column temperature: 40℃, injection volume: 5 μL. Mass spectrometry conditions: ESI ion source in positive ion mode, spray voltage: 3.5 kV, ion transfer tube temperature: 320℃, sheath gas pressure: 35 arb, auxiliary gas pressure: 10 arb. Monitoring was performed using MRM mode, with Q1 and Q3 resolutions both set to 0.7 Da and collision energies of 25-35 eV.

[0008] Example 3: Sample Testing and Result Determination 3.1 Establishment of Standard Curve A stable isotope-labeled internal standard (the internal standard being a YLNDQVAK sequence containing heavily isotope-labeled leucine, such as [13C6,15N2]-leucine-labeled peptide, prepared by solid-phase peptide synthesis) was added to a series of diluted P9 protein standards (concentration range 1-1000 ng / mL). The assay was performed according to the conditions of Example 2, and quantification was performed by peak area ratio, with R² ≥ 0.995.

[0009] 3.2 Sample Testing The enzymatically digested sample prepared in Example 2 was injected and analyzed under the LC-MS / MS conditions described above. The peak areas of the four MRM channels were recorded, and the measured concentration (ng / mL) of P9 protein in the sample was calculated using the internal standard method.

[0010] 3.3 Result Calculation P9 protein content (ng / mg) = (sample concentration × total volume of sample extract) / sample weight.

[0011] 3.4 Standards for Passing and Failure Validity determination: The peak area of ​​the stable isotope-labeled internal standard should be within the expected range (RSD≤15%), and the standard curve R²≥0.995; otherwise, the batch of experiments is invalid.

[0012] Sample judgment: Qualified product (normal P9 protein expression): The signal-to-noise ratio (S / N) of the four characteristic peptides is ≥10, and the calculated measured content of P9 protein is ≥3 ng / mg.

[0013] Non-compliant product (low P9 protein expression): The signal-to-noise ratio (S / N) of the four characteristic peptides is ≥10, but the calculated actual content of P9 protein is <3 ng / mg.

[0014] Seriously non-compliant (P9 protein not detected or below the limit of quantitation): The signal-to-noise ratio (S / N) of any characteristic polypeptide is <3 (judged as "not detected"), or S / N is ≥3 but <10 (judged as "below the limit of quantitation").

[0015] Completely unqualified: If the test sample is found to have both P9 protein and Amuc_1100 protein or Amuc_1409 protein in a "seriously unqualified" state, it will be judged as completely unqualified and will not pass.

Claims

1. A method for quantitative detection of P9 protein in Akkermansia muciniphila, characterized in that, Includes the following steps: (a) Obtain the sample to be tested and preprocess the sample to extract P9 protein; (b) The extracted P9 protein was digested with trypsin to obtain the characteristic polypeptide; (c) The characteristic peptides were detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS); (d) Calculate the content of P9 protein using a standard curve.

2. The detection method according to claim 1, characterized in that, The characteristic polypeptide is a specific polypeptide fragment produced by trypsin digestion of P9 protein, and the characteristic polypeptide is selected from any one or more of the following sequences: (a) YLNDQVAK, with a precursor ion m / z of 472.25 (2+). (b) FVSEAIGK, with a precursor ion m / z of 438.24 (2+); (c) TAAYILGQK, the precursor ion m / z is 501.27 (2+); (d) VVNEALSK, the precursor ion m / z is 452.26 (2+).

3. The detection method according to claim 1, characterized in that, The LC-MS / MS method uses multiple reaction monitoring (MRM) mode for detection, with a collision energy of 20-40 eV.

4. The detection method according to claim 1, characterized in that, The pretreatment includes treating the sample with a lysis buffer containing 8M urea and 10mM DTT, followed by lysis at room temperature for 30 minutes.

5. The detection method according to claim 1, characterized in that, The method also includes quantification using stable isotope-labeled internal standards.

6. The detection method according to claim 1, characterized in that, The linear range of the method is 1-1000 ng / mL, the limit of detection is 0.3 ng / mL, and the limit of quantitation is 1.0 ng / mL.

7. The application of the detection method according to any one of claims 1 to 6 in the quantitative detection of P9 protein in AKK bacteria, strain screening, or product quality control.