Method and kit for quantitatively detecting Amuc1100 protein

Abstract

The invention provides a method and a kit for quantitatively detecting an Amuc1100 protein. The method disclosed by the invention comprises the step of detecting polypeptide of Amuc1100 protein in a sample to be detected through liquid chromatography tandem mass spectrometry, wherein the amino acid sequence of the polypeptide is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. The method disclosed by the invention can be used for stably, accurately and sensitively measuring the content of the Amuc1100 protein in the sample.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and in particular relates to a method and a kit for quantitatively detecting Amuc_1100 protein. Background Art

[0002] The intestinal microbiota is considered a key factor influencing host health, and disturbances in its composition or activity are thought to be associated with the development of numerous diseases (PMID: 28442782). Akkermansia muciniphila (A. muciniphila, or Akkermansia muciniphila) is a newly identified species of the Verrucomicrobiota. It is a Gram-negative, strictly anaerobic bacterium that colonizes the human gastrointestinal mucosa and specifically degrades mucin. It was successfully isolated in 2004 by researchers at the Microbiology Laboratory in Wageningen (PMID: 15388697). Akkermansia muciniphila is ubiquitous in the intestines of healthy adults and infants, comprising 1%-4% of the total intestinal microbiota that colonizes the gut from early life (PMID: 18083887). Recent studies have shown that the absence or reduction of Akkermansia muciniphila is closely associated with a variety of diseases, including obesity, diabetes, hepatic steatosis, inflammation, and response to cancer immunotherapy. In addition, several invention patents have also disclosed the use of Akk bacteria in related diseases, such as the treatment or prevention of diabetes (CN105030841A), obesity (CN105106245A, CN111938158A), and depression (CN110227085A). These studies and invention applications demonstrate that Akk bacteria, as a star of the second-generation probiotics, has potential applications and commercial value in future areas such as food, health products, and medicine.

[0003] With increasing research, the study of Akk bacteria and disease has shifted from correlation to clarifying causal relationships and elucidating mechanistic mechanisms. Not only live Akk bacteria, but also pasteurized Akk bacteria and Akk components, such as outer membrane proteins, secretory proteins, and exosomes, have been shown to play mechanistic roles in disease regulation (PMID: 36835309). Amuc_1100, a protein specific to the outer membrane of Akk bacteria, is one of the specific and unique molecular mechanisms by which Akk bacteria contribute to host health. Recent studies have shown that Amuc_1100 can replicate the beneficial effects of Akk bacteria on diseases, including metabolic disorders, inflammation, and tumors. For example, Amuc_1100 improves metabolism in obese and diabetic mice through the TLR2 pathway (PMID: 27892954). In a mouse colitis model, Amuc_1100 ameliorates colitis by reducing colon-infiltrating macrophages and cytotoxic T lymphocytes (CTLs). In a mouse colon cancer model, Amuc_1100 inhibits ulcerative colitis-associated cancer by inducing TNF-α and inhibiting PD-1 expression, expansion, and activation of CTLs (PMID: 32169907). Amuc_1100 alleviates depressive-like behavior in chronic unpredictable mild stress (CUMS)-induced depressed mice and improves downregulation of brain-derived neurotrophic factor (BDNF) and inflammatory responses in the hippocampus (PMID: 34129964). These findings suggest that Amuc_1100 is a key active substance and mechanistic effector of Akkermansia's probiotic effects on disease.

[0004] Based on the aforementioned considerations regarding the role of Akk bacteria in disease and the impact of its active compound, Amuc_1100, on the potential efficacy of Akk strains in disease, Amuc_1100 can be used as a potential biomarker for evaluating the potential activity and function of Akk bacteria, the efficacy of Akk products, or clinically relevant disease indicators. This is of great significance for the future development, application, and commercialization of Akk bacteria in areas such as food and medicine. Therefore, the identification and quantitative detection of the Amuc_1100 protein is a crucial prerequisite for the functional evaluation of Akk strains and their future application in various scenarios.

[0005] Protein quantification can primarily be achieved through antibodies or mass spectrometry. Currently, the detection of Amuc_1100 protein content in Akk strains has only been reported using antibodies. However, no commercially available Amuc_1100 antibodies or detection kits are available on the market. Most reported antibodies, both domestically and internationally, are produced by research institutions using recombinant Amuc_1100 protein to immunize animals (PMID: 27892954). Animal immunization experiments are significantly affected by the animal's immune response, resulting in low antibody content. Large-scale antibody extraction requires optimized methods. Furthermore, the production of Amuc_1100 antibodies is time-consuming and expensive. These factors currently significantly limit the detection and application of Amuc_1100 protein. Therefore, new methods for the identification and quantification of Amuc_1100 protein are needed. To date, no mass spectrometry methods have been reported for protein identification and quantification in Akk strains or samples containing Akk strains using Amuc_1100 protein signature peptides. Summary of the Invention

[0006] To address the shortcomings of the prior art, the present disclosure provides a method for the accurate, stable, and rapid quantitative detection of the Amuc_1100 protein. The present disclosure has discovered a group of polypeptides that can be used to identify or quantitatively detect the Amuc_1100 protein, enabling stable detection of the protein. Furthermore, peptide standards are synthesized from standard proteins to establish a standard curve for the stable, accurate, and quantitative detection of the Amuc_1100 protein content in test samples.

[0007] According to one aspect of the present disclosure, a method for quantitatively detecting Amuc_1100 protein is provided. The method comprises detecting the polypeptide in the Amuc_1100 protein in a test sample by liquid chromatography tandem mass spectrometry, wherein the amino acid sequence of the polypeptide is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0008] In another aspect of the present disclosure, the polypeptide of Amuc_1100 protein in the test sample is detected by liquid chromatography tandem mass spectrometry, and the characteristic peptides prepared by the standard protein are plotted into a standard curve to facilitate stable and accurate quantitative detection of Amuc-1100 protein in the sample.

[0009] In some embodiments, the quantitative detection comprises the steps of synthesizing a standard of the polypeptide and obtaining a standard curve of the polypeptide based on the mass spectrometry signal intensity of the synthesized standard.

[0010] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 3 is 428-431, for example, 428, 429, 430, 431, or any value therebetween. In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 3 is 429-430, for example, 429.7557.

[0011] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 is 708-711, for example, 708, 709, 710, 711, or any value therebetween. In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 is 709-710, for example, 709.3846.

[0012] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 is 601-604, for example, 601, 602, 603, 604, or any value therebetween. In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 is 602-603, for example, 602.3863.

[0013] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated in the mass spectrometer by the polypeptide having an amino acid sequence as shown in SEQ ID NO: 4 is 557-560, for example, 557, 558, 559, or 560. In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated in the mass spectrometer by the polypeptide having an amino acid sequence as shown in SEQ ID NO: 4 is 558-559, for example, 558.7858.

[0014] In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 3.

[0015] According to another aspect of the present disclosure, a method for quantitatively detecting Amuc_1100 protein is provided, the method comprising detecting a polypeptide of Amuc_1100 protein in a test sample by liquid chromatography tandem mass spectrometry, wherein the amino acid sequence of the polypeptide is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0016] In some embodiments, the quantitative detection comprises the steps of synthesizing a standard of the polypeptide and obtaining a standard curve of the polypeptide based on the mass spectrometry signal intensity of the synthesized standard.

[0017] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 3 is 428-431, for example, 428, 429, 430, 431, or any value therebetween. In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 3 is 429-430, for example, 429.7557.

[0018] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 is 708-711, for example, 708, 709, 710, 711, or any value therebetween. In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 is 709-710, for example, 709.3846.

[0019] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 is 601-604, for example, 601, 602, 603, 604, or any value therebetween. In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the mass spectrometer of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 is 602-603, for example, 602.3863.

[0020] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated in the mass spectrometer by the polypeptide having an amino acid sequence as shown in SEQ ID NO: 4 is 557-560, for example, 557, 558, 559, or 560. In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated in the mass spectrometer by the polypeptide having an amino acid sequence as shown in SEQ ID NO: 4 is 558-559, for example, 558.7858.

[0021] In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 3.

[0022] In some embodiments, in the liquid chromatography tandem mass spectrometry, 0.05-0.2% formic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B. In some specific embodiments, 0.01% formic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B.

[0023] In some embodiments, gradient elution is performed, and the gradient elution program includes: 0-1 min, 90%-80% mobile phase A; 1-1.5 min, 80%-10% mobile phase A; 1.5-3.5 min, 10% mobile phase A; 3.5-3.6 min, 10%-90% mobile phase A; 3.6-5 min, 90% mobile phase A.

[0024] In some embodiments, the chromatography column is a C18 chromatography column.

[0025] In some embodiments, triple quadrupole liquid spectrometry (LC-MS) is used for detection.

[0026] In some embodiments, the method comprises the steps of:

[0027] (1) The sample containing Amuc_1100 protein was enzymatically treated with trypsin and then desalted;

[0028] (2) Detecting the desalted sample by liquid chromatography-tandem mass spectrometry to obtain mass spectrometry data;

[0029] (3) Performing qualitative or quantitative analysis on the Amuc_1100 protein in the test sample based on the obtained mass spectrometry data.

[0030] In some embodiments, in step (1), the sample to be tested contains Akkermansia muciniphila, and the Akkermansia muciniphila is subjected to lysis treatment and bacterial protein extraction, and then subjected to enzymatic hydrolysis and desalting treatment.

[0031] In some embodiments, during the enzymatic hydrolysis treatment, the mass ratio of trypsin to the sample containing the Amuc_1100 protein is 1:(20-100), for example, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100 or any value therebetween.

[0032] In some embodiments, the temperature of the enzymatic hydrolysis treatment is 35-40°C, for example, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any value therebetween.

[0033] In some embodiments, the enzymatic treatment time is 4-24 h, for example, 4 h, 6 h, 8 h, 10 h, 12 h, 16 h, 18 h, 22 h, 24 h or any value thereof.

[0034] In some embodiments, the enzymatic treatment is performed using methods well known in the art.

[0035] In some embodiments, the enzymatic treatment comprises the following steps:

[0036] (1) Dissolve the sample to be tested in a urea-Tris solution (final concentration 5-10 M) to obtain a protein solution;

[0037] (2) Add DTT solution (final concentration of 8-15 mM) to the protein solution for reduction treatment (35-40°C, 30-120 min), and then add IAA solution (final concentration of 20-100 mM) for incubation (room temperature in the dark, 20-90 min);

[0038] (3) After diluting the urea concentration in the sample to be tested to less than 1 M, add trypsin for enzymatic hydrolysis.

[0039] In some embodiments, the desalting treatment is performed using methods well known in the art.

[0040] In some embodiments, the desalting process is performed by solid phase extraction. In some embodiments, the desalting process comprises the following steps:

[0041] (1) The C18 solid phase extraction cartridge was pretreated with methanol and 70% acetonitrile (containing 0.1% formic acid) in sequence;

[0042] (2) The enzymatically treated sample was eluted on a pretreated C18 solid phase extraction column using 0.1% formic acid and 70% acetonitrile (containing 0.1% formic acid);

[0043] (3) The eluate was dried and dissolved in 0.1% formic acid for mass spectrometry detection.

[0044] According to another aspect of the present disclosure, a kit for quantitatively detecting Amuc_1100 protein in a sample to be tested is provided, characterized in that the kit comprises a polypeptide standard, the polypeptide standard comprising at least one polypeptide having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and the quantitative detection further comprises detecting the sample to be tested by liquid chromatography tandem mass spectrometry.

[0045] In some embodiments, the amino acid sequence of the polypeptide standard is shown in SEQ ID NO: 3.

[0046] In some embodiments, the kit further comprises trypsin.

[0047] In some embodiments, the kit further comprises at least one of a urea solution, a DTT solution, and an IAA solution.

[0048] In some embodiments, the kit further comprises at least one of acetone, methanol, and acetonitrile.

[0049] According to another aspect of the present disclosure, provided is a use of the kit described in the present disclosure in the identification and / or quantitative detection of Amuc_1100 protein, wherein the kit detects the polypeptide of the Amuc_1100 protein in the test sample by liquid chromatography tandem mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Shown are the mass spectrometry precursor ion results and secondary spectra of four characteristic peptides of the Amuc_1100 protein according to some embodiments of the present disclosure.

[0051] Figure 2 Shown are the mass spectrometry precursor ion results and secondary spectra of four Amuc_1100 characteristic peptides identified from Akkermansia muciniphila according to some embodiments of the present disclosure.

[0052] Figure 3 A standard curve of characteristic peptides according to some embodiments of the present disclosure is shown.

[0053] Figure 4 Chromatograms and mass spectra of the characteristic peptide AINSLVNK in different samples are shown.

[0054] Figure 5 The technical roadmap of this application is shown. Strain preservation

[0055] The strain Akkermansia muciniphila MNH19250 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with the deposit number GDMCC No: 63782 and the deposit date on June 21, 2024. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The deposit name is Akkermansia muciniphila MNH19250, and the proposed taxonomic name is Akkermansia muciniphila.

[0056] The strain Lactobacillus crispatus MNH22076 was deposited in the Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 65554 and the deposit date on November 28, 2024. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The deposit name is Lactobacillus crispatus MNH22076, and the proposed taxonomic name is Lactobacillus crispatus. DETAILED DESCRIPTION

[0057] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, descriptions of known structures and techniques are omitted in the following description to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and techniques are also described in many publications.

[0058] The reagents and / or kits used in the following examples are all commercially available or can be synthesized by known methods.

[0059] The instruments and reagents involved in the following examples are shown in Tables 1 and 2:

[0060] Table 1

[0061] instrument model brand Ultra-performance liquid chromatography-tandem high-resolution mass spectrometry Thermo Vanquish Nano UHPLC Q-Exactive HF Thermo Fisher Scientific Chromatographic columns Acclaim PepMapTM RSLC (75μm*25cm, nanoViper) Thermo Fisher Scientific High-performance liquid chromatography-tandem high-resolution mass spectrometry 1290-6470B triple quadrupole Agilent Technologies Inc. Chromatographic columns EclipsePlusC18 RRHD (2.1*100mm 1.8μm) Agilent Technologies Inc. analytical balance FA-1004 Shanghai Sunny Hengping Scientific Instrument Co., Ltd. centrifuge TGL-16M Hunan Xiangyi Centrifuge Instrument Co., Ltd.

[0062] Table 2

[0063] Reagents level Manufacturer hydrochloric acid analytically pure Sinopharm Chemical Reagent Co., Ltd. acetone analytically pure Sinopharm Chemical Reagent Co., Ltd. urea analytically pure Aladdin Acetonitrile Chromatographically pure Fisher Scientific Methanol Chromatographically pure Sigma Aldrich Formic acid Mass spectrometry pure Macklin Trypsin Mass spectrometry pure Fisher Scientific water First-class water Fisher Chemical Iodoacetamide (IAA) analytically pure Sigma Aldrich Dithiothreitol (DTT) analytically pure Sigma Aldrich Tris base analytically pure Solarbio

[0064] Example 1: Characteristic peptide identification of Amuc_1100 protein

[0065] In this example, Amuc_1100 recombinant protein was used as a standard sample. The protein was hydrolyzed into peptides by protease, and full scans were performed on a UPLC-QE-MS platform in the data-dependent full-scan (Full MS / data-dependent MS2) mode. The peptides were analyzed using Proteome Discoverer 2.5 software to screen and identify characteristic peptides of the Amuc_1100 protein.

[0066] 1. Sample

[0067] Amuc_1100 recombinant protein, derived from Escherichia coli, was purchased from Wuhan Fine Biotechnology Co., Ltd.

[0068] 2. Experimental Procedure

[0069] 2.1 Protease cleavage

[0070] 50 μg of Amuc_1100 recombinant protein standard was dissolved in 8 M urea (24.0240 g of urea and 0.6057 g of Tris base were dissolved in 40 mL of pure water, the pH was adjusted to 8.0 with hydrochloric acid, and the volume was made up to 50 mL with pure water). DTT (100 mM) was added to the protein solution to a final concentration of 10 mmol / L. The solution was reduced at 37°C for 60 min, and then IAA (100 mM) was added to a final concentration of 50 mM. The solution was incubated at room temperature in the dark for 45 min. Finally, the urea concentration of the sample was diluted to less than 1 M, and trypsin was added at a mass ratio of 1:50 (trypsin: protein). The solution was enzymatically digested at 37°C overnight.

[0071] 2.2 Peptide desalting

[0072] A Waters SEP-PAK C18 solid-phase extraction cartridge was placed in a 15 mL centrifuge tube, 1 mL of methanol was added, and the mixture was centrifuged at 150 r / min for 1 min. 0.5 mL of 70% acetonitrile (containing 0.1% formic acid) was added, and the mixture was centrifuged at 150 r / min for 1 min. The enzymatically digested sample was loaded, centrifuged at 150 r / min for 3 min, 1 mL of 0.1% formic acid was added, and the mixture was centrifuged at 150 r / min for 3 min, which was repeated three times. The solid-phase extraction cartridge was removed and placed in a new 15 mL centrifuge tube, 0.5 mL of 70% acetonitrile (containing 0.1% formic acid) was added, and the mixture was centrifuged at 150 r / min for 3 min, which was repeated once. The eluent was the desalted peptide fragment, which was blown dry, dissolved in 100 μL of 0.1% formic acid, and centrifuged at 10,000 r / min for 10 min. The supernatant was lyophilized and subsequently subjected to mass spectrometry detection.

[0073] 2.3 Mass spectrometry detection of characteristic peptides

[0074] Lyophilized samples were dissolved in 0.1% formic acid in water and separated using a UPLC system. After separation by the UPLC system, the peptides were injected into the ESI ion source for ionization and then analyzed by a Q-Exactive HF mass spectrometer. Both the peptide precursor ions and their secondary fragments were detected using the high-resolution Orbitrap probe within the Q-Exactive HF. The conditions and parameters for UPLC separation and mass spectrometry are shown in Table 3.

[0075] Table 3

[0076]

[0077] The mass spectrometry data were analyzed using Proteome Discoverer 2.5 software to identify and screen the characteristic peptides. The peptides containing mis-cleavage or missed cleavage sites, glycosylation motifs containing methionine (M, easily oxidized), NXS / T, and peptide sequences that were easily cyclized and easily oxidized were excluded to obtain the characteristic peptides of the target protein, as shown in Table 4.

[0078] Table 4. Identification of characteristic peptides of Amuc_1100 recombinant protein

[0079]

[0080] The test results are shown in Table 4. By performing enzymatic hydrolysis and mass spectrometry on the Amuc_1100 protein standard, four highly sensitive characteristic peptides of the Amuc_1100 protein were identified. The corresponding parent ion results and secondary spectra of the peptides are shown in Table 4. Figure 1 , among which EQVFVQVSLNLVHFNQPK [m / z 709.3846 (z=3)]; ISIAAK [m / z 602.3863 (z=1)]; AINSLVNK [m / z 429.7557 (z=2)]; SANAAEITPSR [m / z 558.7858 (z=2)].

[0081] Example 2: Identification and Verification of the Amuc_1100 Characteristic Peptide in Akkermansia muciniphila

[0082] In this example, Akkermansia muciniphila was used as a real sample, and the characteristic peptides of the Amuc_1100 protein in the bacteria were identified by mass spectrometry to verify the specificity of the four characteristic peptides identified in Example 1.

[0083] 1. Sample

[0084] The strain used in this experiment was Akkermansia muciniphila MNH19250, which was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposited name of Akkermansia muciniphila MNH19250 and the deposited number of the strain being GDMCC No: 63782.

[0085] 2. Experimental Procedure

[0086] 2.1 Cultivation

[0087] MNH19250 seeds were inoculated into activation medium (medium 1, the formula is shown in Table 5) for activation, cultured anaerobically at 37°C for 24-36 h (standard OD600 ≥ 1.6), and then subcultured into medium 2 (the formula is shown in Table 6) at a ratio of 2.5%, cultured anaerobically at 37°C for 48 h, and the bacterial suspension was collected for subsequent experiments.

[0088] Table 5

[0089]

[0090] Table 6

[0091]

[0092] 2.2 Characteristic peptide detection

[0093] 2.2.1 Protein extraction and enzyme digestion

[0094] A suspension of Akkermansia muciniphila (3.5-4 ml) was centrifuged and the supernatant discarded. 8 M urea was added to the pellet, disrupted by sonication, extracted, and centrifuged at 12,000 rpm for 20 minutes. The supernatant was removed and precipitated with acetone. The resulting protein precipitate was washed three times with acetone, air-dried, and reconstituted with 8 M urea solution. DTT was added to a final concentration of 10 mM and reduced at 37°C for 60 minutes. IAA was then added to a final concentration of 50 mM and incubated at room temperature in the dark for 45 minutes. Finally, the sample was diluted to a urea concentration below 1 M. Trypsin was added at a mass ratio of 1:50 (trypsin: protein precipitate) and digested at 37°C overnight.

[0095] 2.2.2 Peptide desalting

[0096] The method of this step is the same as the peptide desalting treatment in Example 1.

[0097] 2.2.3 Mass spectrometry detection of characteristic peptides

[0098] The mass spectrometry detection method is the same as that in Example 1. The detection results are shown in Table 7:

[0099] Table 7. Verification of characteristic peptides of Amuc_1100 recombinant protein in MNH19250

[0100]

[0101] The four characteristic peptides of Amuc_1100 identified in Example 1: SANAAEITPSR, ISIAAK, AINSLVNK, EQVFVQVSLNLVHFNQPK can be identified in MNH19250. Figure 2 , the corresponding precursor ion results and secondary spectra of the peptide fragments, including; SANAAEITPSR [m / z 558.7891 (z=2)]; ISIAAK [m / z 602.3925 (z=1)]; AINSLVNK [m / z 429.7571 (z=2)]; EQVFVQVSLNLVHFNQPK [m / z 709.3846 (z=3)].

[0102] Example 3: Method for quantitative detection of Amuc_1100 protein using liquid chromatography tandem mass spectrometry

[0103] The principle of quantitative detection of Amuc_1100 protein is as follows: first, based on the four Amuc_1100 characteristic peptides identified in Example 1, further screening is performed based on the principles of peptide stability and high mass spectrometry detection sensitivity. Then, based on the mass spectrometry screening results and software simulation, the characteristic peptide AINSLVNK is selected as the characteristic peptide for quantitative detection of Amuc_1100 protein. A standard curve is established using peptide standards for quantification of the Amuc_1100 protein content in AKK bacteria or real samples.

[0104] 1. Sample

[0105] AINSLVNK peptide standard, 98% purity, molecular weight 858.50434. Synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0106] 2. Experimental Procedure

[0107] 2.1 Reagent Configuration

[0108] 1) Peptide standard stock solution

[0109] Accurately weigh 5.00 mg of the synthetic peptide and place it in a 5 mL volumetric flask. Dissolve it in 0.1% formic acid aqueous solution and dilute to the mark. Shake well to prepare a stock solution with a concentration of 1000 μg / mL.

[0110] 2) Linear solution

[0111] Accurately measure 0.1 mL of the peptide standard stock solution and place it in a 10 mL volumetric flask. Add 0.1% formic acid and dilute to the mark. Shake well to obtain a 10 mg / L standard working solution. Dilute it 10-fold to obtain 1 mg / L. Take the prepared 1 mg / L standard working solution and dilute it sequentially to obtain 500, 100, 50, 10, 5, and 1 μg / L standard working solutions. These will serve as the peptide standard linear working dilutions.

[0112] 2.2 Standard curve drawing

[0113] The characteristic peptide content in the linear working dilution of the peptide standard prepared above (500, 100, 50, 10, 5, 1 μg / L) was detected by triple quadrupole liquid chromatography-mass spectrometry, and a standard curve was drawn. The standard curve was then used to quantify the characteristic peptide content in the real sample and finally convert it into the content of Amuc_1100 protein.

[0114] The HPLC-MS / MS detection conditions are shown in Table 8 below.

[0115] Table 8

[0116]

[0117] The results of the standard curve test are shown in Table 9 and Figure 3 As shown in the results, the mass spectrometry detected the standard sample of the characteristic peptide concentration, and the linear equation was fitted according to the peptide concentration (ng / ml) and the response (Responses) of the peptide detected by mass spectrometry. The formula is: y = 424.313931 × x - 74.551784, R 2 = 0.99961056, indicating a linear relationship between the peptide response signal and peptide concentration, and that the concentration detected by mass spectrometry deviates from the standard average concentration by approximately 10%. Therefore, this standard curve can be used to quantify this characteristic peptide in real samples and ultimately calculate the Amuc_1100 protein content of the sample.

[0118] Table 9 Standard curve detection results

[0119]

[0120] Example 4: Quantitative detection of Amuc_1100 protein in AKK bacteria

[0121] To evaluate the ability of the Amuc_1100 protein quantitative detection method established in Example 3 to detect Amuc_1100 content in real samples, two different Akkermansia samples were prepared: a conventionally cultured Akkermansia suspension and an Akkermansia sample cultured with Garcinia cambogia extract (purchased from Changsha Huirui Biotechnology Co., Ltd.). (Garcinia cambogia is insoluble in the culture medium, increasing the complexity of the bacterial slurry sample.) Furthermore, Lactobacillus crispatus (MNH22076) was used as a negative control. The established Amuc_1100 quantitative method was used to detect the content of real samples.

[0122] 1. Sample

[0123] 1) strain

[0124] Akkermansia muciniphila is MNH19250 in Example 2; Lactobacillus crispatus is Lactobacillus crispatus MNH22076, which are deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number of GDMCC No: 65554.

[0125] 2) Bacteria detection sample preparation

[0126] MNH19250 conventional culture suspension sample (MNH19250 bacterial suspension): According to the conditions of Example 2, the seeds were activated and inoculated into 100 mL of culture medium. The culture was carried out under anaerobic conditions at 37°C for 48 h. When the OD600 value was 2.03, the bacterial suspension was collected.

[0127] MNH19250 fermentation culture sludge sample (MNH19250 sludge): Based on culture medium 2, Garcinia cambogia extract (3 g / L) was added to prepare the fermentation medium. After the seeds were activated, they were inoculated into 1 L of fermentation culture and cultured under anaerobic conditions at 37°C for 48 h. OD600 = 2.26. Then, the supernatant was removed by centrifugation and the sludge sediment was collected.

[0128] MNH22076 bacterial suspension sample (MNH22076 bacterial solution): According to the conditions of Example 2, the seeds were activated and inoculated into 100 mL of culture medium. The culture was carried out under anaerobic conditions at 37°C for 14 h. When OD600 was 2.21, the bacterial suspension was collected.

[0129] 2. Experimental Procedure

[0130] 2.1 Protein extraction

[0131] 1) Bacterial suspension sample: Take an appropriate amount of sample (3.5-4 ml), centrifuge, discard the supernatant, add 8 M urea to the pellet, ultrasonically disrupt, extract, and centrifuge at 12,000 rpm for 20 min. Remove the supernatant and bring the volume up to 500 μL. A small amount of the supernatant is used for protein concentration determination using the Bradford method (Coomassie Brilliant Blue). The remaining supernatant is precipitated with acetone. The resulting protein pellet is washed three times with acetone, air-dried, and reconstituted with 8 M urea solution. DTT is added to a final concentration of 10 mM and reduced at 37°C for 60 min. IAA is then added to a final concentration of 50 mM. Incubate at room temperature in the dark for 45 min. Finally, the sample is diluted to a urea concentration below 1 M. Trypsin is added at a 1:50 mass ratio (trypsin:protein) and digested at 37°C overnight.

[0132] 2) Sludge Sample: Take an appropriate amount of sample (approximately 0.5 g) and add 8 M urea for ultrasonic disruption and extraction. Centrifuge at 12,000 rpm for 20 minutes. Collect the supernatant and bring the volume up to 500 μL. A small amount of the supernatant is used for protein concentration analysis. The remaining supernatant is precipitated with acetone. The resulting protein precipitate is washed three times with acetone, air-dried, and reconstituted with 8 M urea solution. DTT is added to the protein solution to a final concentration of 10 mM and reduced at 37°C for 60 minutes. IAA is then added to a final concentration of 50 mM and incubated at room temperature in the dark for 45 minutes. Finally, the sample is diluted to a urea concentration below 1 M. Trypsin is added at a 1:50 mass ratio (trypsin:protein) and digested at 37°C overnight.

[0133] 3) Total protein content of samples (Bradford method)

[0134] (1) Preparation of Bradford stain: Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol, add 100 ml of 85% phosphoric acid, and then make up to 200 ml with distilled water. This stain remains stable at 4°C for at least 6 months.

[0135] (2) Preparation of protein samples for standard curve: Prepare a set of BSA solutions with concentrations of 0.10 mg / ml, 0.08 mg / ml, 0.06 mg / ml, 0.04 mg / ml, and 0.02 mg / ml for standard curve.

[0136] (3) Dissolve the sample to be tested in a buffer solution that should be the same as the buffer solution used to prepare the standard curve (PBS is best).

[0137] (4) Dilute the concentrated dye binding solution with distilled water at a ratio of 1:4. If a precipitate appears, remove it by filtration.

[0138] (5) Add 5 ml of the diluted dye binding solution to each sample and allow to react for 5 to 30 minutes. The dye will change from red to blue after binding to the protein. Measure its absorbance at a wavelength of 595 nm. Note that the color reaction should not exceed 30 minutes.

[0139] (6) Calculate the protein concentration of the sample to be tested based on the standard curve, and finally calculate the total protein content by the protein concentration and the volume of the sample protein extract.

[0140] 2.2 Peptide desalting

[0141] The peptide desalting method was the same as that in Example 1.

[0142] 2.3 Quantitative detection of Amuc_1100 protein in samples

[0143] Referring to the method in Example 3, a standard curve was drawn using characteristic peptide standards, and then the characteristic peptide content in the sample was detected using a triple quadrupole liquid chromatography-mass spectrometry instrument. Figure 4 The middle ones are the chromatograms of different samples and the mass spectrum of the characteristic peptide AINSLVNK.

[0144] 2.4 Calculation method of test sample content

[0145] 1) The calculation formula can be used to convert the Amuc_1100 content in liquid samples or solid samples. The formula is as follows:

[0146] The content of Amuc_1100 in the test sample (ng / mg or ng / mL) = (CX-C0) × M1 × V / m / M2 / 1000 (Formula 1)

[0147] m: liquid (volume of sample, in liters (L)) or solid (mass of sample, in grams (g));

[0148] M1: protein molecular weight (target protein molecular weight), Amuc_1100 protein is 34213 Da;

[0149] M2: molecular mass of peptide standard (characteristic peptide molecular weight), the peptide is 858.50434 Da;

[0150] V: Final volume of the sample solution, in milliliters (mL), which refers to the final volume of the desalted lyophilized peptide, i.e. 0.1 mL;

[0151] C0: mass concentration of blank, in micrograms per liter (μg / L);

[0152] CX: The peptide concentration of the test substance, expressed in micrograms per liter (μg / L).

[0153] 2) Amuc_1100 protein content per mg of total bacterial protein (ng / mg) = Amuc_1100 content in sample / total bacterial protein (Formula 2)

[0154] 2.5 Sample Amuc_1100 Test Results and Analysis

[0155] Quantitative analysis of protein extracts from three samples of MNH19250 bacterial suspension, MNH19250 bacterial sludge, and MNH22076 bacterial suspension revealed total bacterial protein contents of 0.43 mg, 1.30 mg, and 0.71 mg, respectively. The results in Table 10 show that the mass spectrometry analysis of the characteristic peptide of the Amuc_1100 protein in these samples revealed concentrations of 5.68 ng / mL, 21.18 ng / mL, and 0 ng / mL. Finally, the Amuc_1100 protein content in each sample was calculated using Equation 1 to be 5.66 ng / mL, 0.16 ng / mg, and 0 ng / mg (not detected). In addition, the Amuc_1100 protein content per milligram of bacterial protein was calculated using Equation 2, yielding 52.65 ng of Amuc_1100 protein per milligram of bacterial protein in the AKK bacterial liquid sample and 64.6 ng of Amuc_1100 protein per milligram of bacterial protein in the AKK bacterial sludge sample. The results demonstrated that the established quantitative detection method failed to detect Amuc_1100 in the negative control strain Lactobacillus crispatus, while the Amuc_1100 protein content was quantified in Akkermansia muciniphila prepared by the two different methods, with similar levels, validating the accuracy of the detection method.

[0156] Table 10

[0157]

[0158] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of a polypeptide in the quantitative detection of Amuc_1100 protein, characterized in that: The quantitative detection is performed by liquid chromatography tandem mass spectrometry to detect the polypeptide of the Amuc_1100 protein in the test sample containing the bacterial liquid or sludge of Akkermansia muciniphila, and the amino acid sequence of the polypeptide is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

2. The use according to claim 1, characterized in that The quantitative detection comprises synthesizing a standard substance of the polypeptide, and obtaining a standard curve of the polypeptide according to the mass spectrometry signal intensity of the synthesized standard substance.

3. The use according to claim 1, characterized in that The mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide having the amino acid sequence shown in SEQ ID NO: 3 in the mass spectrometer is 428-431; and / or The mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 in the mass spectrometer is 708-711; and / or The mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide having the amino acid sequence shown in SEQ ID NO: 2 in the mass spectrometer is 601-604; and / or The mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide with the amino acid sequence shown in SEQ ID NO: 4 in the mass spectrometer is 557-560.

4. A method for quantitatively detecting Amuc_1100 protein, characterized in that: The method includes detecting a polypeptide of the Amuc_1100 protein in a test sample containing a bacterial liquid or sludge of Akkermansia muciniphila by liquid chromatography tandem mass spectrometry, wherein the amino acid sequence of the polypeptide is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

5. The method according to claim 4, characterized in that The quantitative detection comprises the steps of synthesizing a standard substance of the polypeptide and obtaining a standard curve of the polypeptide according to the mass spectrometry signal intensity of the synthesized standard substance.

6. The method according to claim 4, characterized in that In the liquid chromatography tandem mass spectrometry, 0.05-0.2% formic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is performed. The gradient elution program includes: 0-1 min, 90%-80% mobile phase A; 1-1.5 min, 80%-10% mobile phase A; 1.5-3.5 min, 10% mobile phase A; 3.5-3.6 min, 10%-90% mobile phase A; 3.6-5 min, 90% mobile phase A; and / or The chromatographic column is a C18 column; and / or Detection was performed using a triple quadrupole liquid chromatography-mass spectrometry instrument.

7. The method according to claim 4, characterized in that The method comprises the following steps: (1) The sample containing Amuc_1100 protein was enzymatically treated with trypsin and then desalted; (2) Detecting the desalted sample by liquid chromatography-tandem mass spectrometry to obtain mass spectrometry data; (3) Performing qualitative or quantitative analysis on the Amuc_1100 protein in the test sample based on the obtained mass spectrometry data.

8. The method according to claim 7, characterized in that In step (1), the sample to be tested contains Akkermansia muciniphila, and the Akkermansia muciniphila is subjected to a lysis treatment and bacterial protein is extracted, followed by an enzymatic hydrolysis treatment and a desalting treatment; and / or In the enzymatic hydrolysis treatment, the mass ratio of trypsin to the sample containing the Amuc_1100 protein is 1:(20-100); and / or the temperature of the enzymatic hydrolysis treatment is 35-40° C.; and / or the time of the enzymatic hydrolysis treatment is 4-24 hours.

9. The method according to claim 4, characterized in that The mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide having the amino acid sequence shown in SEQ ID NO: 3 in the mass spectrometer is 428-431; and / or The mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 in the mass spectrometer is 708-711; and / or The mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide having the amino acid sequence shown in SEQ ID NO: 2 in the mass spectrometer is 601-604; and / or The mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide with the amino acid sequence shown in SEQ ID NO: 4 in the mass spectrometer is 557-560.

10. A kit for identifying and / or quantitatively detecting Amuc_1100 protein from a bacterial liquid or sludge containing Akkermansia muciniphila, characterized in that: The kit includes a polypeptide standard, which includes at least one polypeptide with an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4. The quantitative detection further includes detecting a test sample containing Akkermansia muciniphila bacterial liquid or sludge by liquid chromatography tandem mass spectrometry.

Images