Characterized polypeptide of akkermansia muciniphila amuc_1409 protein and lc-ms / ms quantitative detection method

Abstract

The present application relates to the characteristics of mucinophilic akkermansia Amuc_1409 protein polypeptide and LC-MS / MS quantitative detection method.The characteristic polypeptide is specific polypeptide fragment produced by trypsin enzymolysis of Amuc_1409 protein, selected from AQSFLENK, YVSDGVLK or FGENATR.The LC-MS / MS detection method provided by the present application can realize high-precision quantitative detection of AKK bacteria Amuc_1409 protein, and the linear range is 1-1000 ng / mL.The actual content of Amuc_1409 protein is greater than or equal to 3 ng / mg, which is qualified.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to the characteristic polypeptides of Akkermansia muciniphila Amuc_1409 protein and a quantitative detection method by LC-MS / MS. Background Technology

[0002] Amuc_1409 is a core functional protein in *Amycosis ulcerans* (AKK) related to intestinal barrier repair and immune regulation, and is closely related to the maintenance of intestinal barrier integrity and the regulation of immune homeostasis. Literature (e.g., Derrien M. et al., *Int J Syst Evol Microbiol*, 2004) has confirmed that *Amycosis ulcerans* plays a crucial role in maintaining intestinal epithelial barrier function and regulating immune homeostasis, and Amuc_1409 is one of the key functional proteins in this pathway. The content of this protein is an important indicator for evaluating the intestinal barrier repair activity of *Amycosis ulcerans* products.

[0003] Existing methods for detecting Amuc_1409 protein primarily rely on immunological detection. However, immunological detection depends on the availability of specific antibodies, and batch-to-batch antibody variability can affect the accuracy of the results. Mass spectrometry (LC-MS / MS), as a high-precision quantitative method, is antibody-independent and offers higher specificity and accuracy. However, no LC-MS / MS quantitative detection method based on the characteristic peptides of Amuc_1409 protein has yet been established in the current technology. Summary of the Invention

[0004] ① Problem to be solved: To provide an antibody-independent, high-precision, and highly specific method for the quantitative detection of Amuc_1409 protein, so as to achieve accurate quantification of Amuc_1409 protein in AKK bacteria products.

[0005] ② Technical solution: This invention provides three characteristic peptides (AQSFLENK, YVSDGVLK, FGENATR) generated after trypsin digestion of Amuc_1409 protein, and an LC-MS / MS quantitative detection method based on these characteristic peptides, with a linear range of 1-1000 ng / mL and a detection limit of 0.3 ng / mL.

[0006] 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

[0007] Example 1: Screening and identification of characteristic peptides of Amuc_1409 protein Recombinant Amuc_1409 protein (theoretical molecular weight approximately 29.5 kDa) was digested with trypsin, and the digestion products were analyzed by LC-MS / MS. By comparing the amino acid sequence of Amuc_1409 protein with mass spectrometry data, the following three characteristic polypeptide sequences were identified: Peptide number sequence, precursor ion m / z, retention time (min), specificity A1409-P1 AQSFLENK 465.24 (2+) 10.8 Amuc_1409 specific A1409-P2 YVSDGVLK 442.25 (2+) 12.3 Amuc_1409 specific A1409-P3 FGENATR 412.21 (2+) 9.5 Amuc_1409 specific Example 2: Sample pretreatment and enzymatic digestion Take 100 mg of AKK lyophilized powder and add 1 mL of extraction buffer (8 M urea, 50 mM NH4HCO3, 10 mM DTT). Extract at room temperature with shaking for 2 hours, then centrifuge at 12,000 g for 20 minutes at 4 °C 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 incubate overnight (16 hours) at 37 °C. Desalt the enzymatic hydrolysate using a C18 solid-phase extraction column, freeze-dry, and reconstitute in 100 μL of 0.1% formic acid aqueous solution.

[0008] Example 3: Sample Testing and Result Determination 3.1 Instrument Conditions The liquid chromatography system was UHPLC, and the column was an ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Gradient elution program: 0–2 min 5% B, 2–25 min 5–40% B, 25–26 min 40–95% B, 26–28 min 95% B, 28–28.5 min 95–5% B, 28.5–32 min 5% B. Flow rate: 0.3 mL / min, column temperature: 40 °C, injection volume: 5 μL.

[0009] 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 in MRM mode, with Q1 and Q3 resolutions both set to 0.7 Da and collision energies of 25-35 eV.

[0010] 3.2 Establishment of Standard Curve Add the stable isotope-labeled internal standard to a series of diluted Amuc_1409 protein standard (concentration range 1-1000 ng / mL), and detect under the above conditions. Quantify by peak area ratio, and R² should be ≥0.995.

[0011] 3.3 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 three MRM channels were recorded, and the measured concentration (ng / mL) of Amuc_1409 protein in the sample was calculated using the internal standard method.

[0012] 3.4 Result Calculation Amuc_1409 protein content (ng / mg) = (sample concentration × total volume of sample extract) / sample weight.

[0013] 3.5 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.

[0014] Sample judgment: Qualified products (normal Amuc_1409 protein expression): must simultaneously meet the following requirements: (1) The signal-to-noise ratio (S / N) of the three characteristic peptides is ≥10; (2) The calculated Amuc_1409 protein content is ≥ 3 ng / mg.

[0015] Non-compliant product (low expression of Amuc_1409 protein): The signal-to-noise ratio (S / N) of the three characteristic peptides is ≥10, but the calculated measured content of Amuc_1409 protein is <3 ng / mg.

[0016] Serious non-compliance (Amuc_1409 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").

[0017] Completely unqualified: If the test sample is found to have Amuc_1409 protein and P9 protein or Amuc_1100 protein both showing "serious unqualified" conditions, it is judged as completely unqualified and will not pass.

Claims

1. A characteristic polypeptide for the detection of proteins in Akkermansia muciniphila Amuc_1409, characterized in that, The characteristic polypeptide is a specific polypeptide fragment produced by trypsin digestion of Amuc_1409 protein.

2. The characteristic polypeptide according to claim 1, characterized in that, The characteristic polypeptide is selected from any one or more of the following sequences: (a) AQSFLENK, with a precursor ion m / z of 465.24 (2+). (b) YVSDGVLK, with a parent ion m / z of 442.25 (2+). (c) FGENATR, with a precursor ion m / z of 412.21 (2+).

3. The application of the characteristic polypeptide according to claim 1 or 2 in the quantitative detection of Amuc_1409 protein in AKK bacteria by LC-MS / MS.

4. A method for quantitative detection of Amuc_1409 protein in AKK bacteria by LC-MS / MS, characterized in that, Includes the following steps: (a) Obtain the sample to be tested and preprocess the sample to extract Amuc_1409 protein; (b) The extracted Amuc_1409 protein was digested with trypsin to obtain the characteristic polypeptide of claim 1; (c) The characteristic peptides were detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS); (d) The content of Amuc_1409 protein was calculated using a standard curve.

5. The detection method according to claim 4, characterized in that, The LC-MS / MS method uses multiple reaction monitoring (MRM) mode for detection, with a collision energy of 20-35 eV.

6. The detection method according to claim 4, 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 4 to 6 in the quantitative detection of protein in AKK strain Amuc_1409, strain screening, or product quality control.