Method and kit for quantitatively detecting Amuc_1100 protein

Abstract

The present disclosure provides a method and a kit for quantitatively detecting Amuc_1100 protein. The method of the present disclosure includes detecting polypeptides of Amuc_1100 protein in a sample to be tested by liquid chromatography tandem mass spectrometry, 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. The method of the present disclosure can stably, accurately and sensitively measure the content of Amuc_1100 protein in a sample.

Description

Technical Field

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

[0002] The gut microbiota is considered to be one of the key factors affecting host health, and disorders in its composition or activity are considered to be related to the occurrence of various diseases (PMID: 28442782). Akkermansia muciniphila ( Akkermansia muciniphila , hereinafter referred to as A. muciniphila or Akk bacterium), is a new species of a new genus in the phylum Verrucomicrobiota. It colonizes the human digestive gastrointestinal mucosa and is a Gram-negative strict anaerobe that can specifically degrade mucin. It was successfully isolated by researchers at the Microbiology Laboratory in Wageningen in 2004 (PMID: 15388697). Akk bacteria are prevalent in the intestines of healthy adults and infants, accounting for 1% - 4% of the total gut microbiota colonized from early life (PMID: 18083887). Recent studies have found that the lack or reduction of Akk bacterium colonization is closely related to various diseases, such as obesity, diabetes, hepatic steatosis, inflammation, and the response to cancer immunotherapy. In addition, some invention patents have also disclosed the application of Akk bacteria in related diseases, such as the use of Akk bacteria in the treatment or prevention of diabetes (CN105030841A), obesity (CN105106245A, CN111938158A), depression (CN110227085A), etc. These research and invention applications indicate that as a star of the second-generation probiotics, Akk bacteria have potential applications and commercial value in the fields of future food, health products, medicine, etc.

[0003] With the in-depth research, the study on the relationship between Akkermansia muciniphila and diseases has shifted from correlation issues to the analysis of clear causal relationships and mechanism functions. Not only live Akkermansia muciniphila, but also pasteurized Akkermansia muciniphila and its components, such as outer membrane proteins, secreted proteins, and extracellular vesicles, have been proven to play regulatory roles in diseases (PMID: 36835309). Amuc_1100 is a specific protein in the outer membrane of Akkermansia muciniphila. As one of the specific and unique molecular mechanisms by which Akkermansia muciniphila acts on host health, recent studies have found that Amuc_1100 can reproduce the beneficial effects of Akkermansia muciniphila on diseases (such as metabolic diseases, inflammation, and tumors). For example, Amuc_1100 improves the metabolism of obese and diabetic mice through the TLR2 pathway (PMID: 27892954); in a mouse colitis model, Amuc_1100 can improve colitis by reducing infiltrated macrophages and cytotoxic T lymphocytes (CTLs) in the colon, while in a mouse colon cancer model, Amuc_1100 can inhibit ulcerative colitis-associated cancer by inducing TNF-α and inhibiting PD-1 expression, proliferation, and activation of CTLs (PMID: 32169907); Amuc_1100 can relieve depressive-like behaviors in mice with depression induced by chronic unpredictable mild stress (CUMS), and improve the downregulation of brain-derived neurotrophic factor (BDNF) and inflammatory responses in the hippocampal region (PMID: 34129964). These evidences indicate that the Amuc_1100 protein is an important active substance and mechanism effector for Akkermansia muciniphila to exert probiotic effects on diseases.

[0004] Based on the above considerations regarding the role of Akkermansia muciniphila in diseases and the impact of its active substance Amuc_1100 on the potential efficacy of Akkermansia muciniphila strains in diseases, Amuc_1100 can be used as one of the potential evaluation active markers for the potential activity and function of Akkermansia muciniphila, or the efficacy of Akkermansia muciniphila products, or clinical-related disease indicators. It is of great significance for the future development, application, and commercialization of Akkermansia muciniphila in fields including food and medicine. Therefore, the identification and quantitative detection of the Amuc_1100 protein are important prerequisites for the functional evaluation of Akkermansia muciniphila strains or their future applications in different scenarios.

[0005] For protein quantitative detection, it can be mainly achieved by antibody or mass spectrometry methods. Currently, the detection of the protein content of Amuc_1100 in Akk strain has only been reported to be achieved by antibody methods. However, there are no commercially available antibodies or detection kits for Amuc_1100 protein on the current market. Most of the antibody methods reported at home and abroad are prepared by scientific research institutions themselves through the method of immunizing animals with Amuc_1100 recombinant protein (PMID: 27892954). Animal immunization experiments are greatly affected by animal immune responses, resulting in a small amount of antibodies obtained. The method needs to be optimized for large-scale antibody extraction. In addition, the production of Amuc_1100 antibodies also involves long time and cost issues. These reasons currently greatly limit the detection and application of Amuc_1100 protein. Therefore, there is a need to develop a new method for identifying and quantifying Amuc_1100 protein. So far, there has been no literature report on the mass spectrometry method for identifying and quantitatively detecting Akk strain or samples containing Akk strain using the characteristic peptides of Amuc_1100 protein. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present disclosure provides a method for accurately, stably and rapidly quantitatively detecting Amuc_1100 protein. The present disclosure has discovered a group of polypeptides that can be used for identifying or quantitatively detecting Amuc_1100 protein, and such polypeptides can achieve stable detection of Amuc_1100 protein. Further, by synthesizing polypeptide standards from standard proteins, a standard curve is established for stably and accurately quantitatively detecting the content of Amuc_1100 protein in the sample to be tested.

[0007] According to one aspect of the present disclosure, there is provided an application of a polypeptide in quantitatively detecting Amuc_1100 protein, wherein the quantitative detection detects the polypeptide in Amuc_1100 protein in the sample to be tested by liquid chromatography-tandem mass spectrometry, 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.

[0008] In another aspect of the present disclosure, the polypeptide of Amuc_1100 protein in the sample to be tested is detected by liquid chromatography-tandem mass spectrometry, and a standard curve is drawn by the characteristic peptides prepared from standard proteins, so as to stably and accurately quantitatively detect the Amuc-1100 protein in the sample.

[0009] In some embodiments, the quantitative detection includes the steps of synthesizing a standard product of the polypeptide and obtaining a standard curve of the polypeptide according to the mass spectrometry signal intensity of the synthesized standard product.

[0010] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide with the amino acid sequence as shown in SEQ ID NO: 3 in mass spectrometry is 428 - 431, such as 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 polypeptide with the amino acid sequence as shown in SEQ ID NO: 3 in mass spectrometry is 429 - 430, such as 429.7557.

[0011] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide with the amino acid sequence as shown in SEQ ID NO: 1 in mass spectrometry is 708 - 711, such as 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 polypeptide with the amino acid sequence as shown in SEQ ID NO: 1 in mass spectrometry is 709 - 710, such as 709.3846.

[0012] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide with the amino acid sequence as shown in SEQ ID NO: 2 in mass spectrometry is 601 - 604, such as 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 polypeptide with the amino acid sequence as shown in SEQ ID NO: 2 in mass spectrometry is 602 - 603, such as 602.3863.

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

[0014] In some embodiments, the amino acid sequence of the polypeptide is as 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 includes detecting the polypeptide of Amuc_1100 protein in a sample to be tested by liquid chromatography tandem mass spectrometry, 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.

[0016] In some embodiments, the quantitative detection includes synthesizing a standard of the polypeptide and obtaining a standard curve of the polypeptide according to 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 polypeptide with the amino acid sequence shown in SEQ ID NO: 3 in mass spectrometry is 428 - 431, such as 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 polypeptide with the amino acid sequence shown in SEQ ID NO: 3 in mass spectrometry is 429 - 430, such as 429.7557.

[0018] In some embodiments, 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: 1 in mass spectrometry is 708 - 711, such as 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 polypeptide with the amino acid sequence shown in SEQ ID NO: 1 in mass spectrometry is 709 - 710, such as 709.3846.

[0019] In some embodiments, 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: 2 in mass spectrometry is 601 - 604, such as 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 polypeptide with the amino acid sequence shown in SEQ ID NO: 2 in mass spectrometry is 602 - 603, such as 602.3863.

[0020] In some embodiments, 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 mass spectrometry is 557 - 560, such as 557, 558, 559, 560. In some embodiments, 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 mass spectrometry is 558 - 559, such as 558.7858.

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

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

[0023] In some embodiments, gradient elution is used for elution, and the program of the gradient elution 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 chromatographic column is a C18 chromatographic column.

[0025] In some embodiments, a triple quadrupole liquid chromatography - mass spectrometry instrument is used for detection.

[0026] In some embodiments, the method includes the following steps:

[0027] (1) Using trypsin to enzymatically digest the sample to be tested containing Amuc_1100 protein, and then performing desalting treatment;

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

[0029] (3) Qualitatively or quantitatively analyzing the Amuc_1100 protein in the sample to be tested according to the obtained mass spectrometry data.

[0030] In some embodiments, in step (1), the sample to be tested contains Akkermansia muciniphila. After lysing the Akkermansia muciniphila and extracting bacterial proteins, enzymatic digestion treatment and desalting treatment are then carried out.

[0031] In some embodiments, in the enzymatic digestion treatment, the mass ratio of trypsin to the sample to be tested containing 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 digestion 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 time of the enzymatic digestion treatment 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 therebetween.

[0034] In some embodiments, the enzymatic digestion treatment is carried out by a method well - known in the art.

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

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

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

[0038] (3) Dilute the urea concentration in the sample to be tested to less than 1 M, and then add trypsin for enzymatic digestion.

[0039] In some embodiments, the desalting treatment is carried out by a method well-known in the art.

[0040] In some embodiments, the desalting treatment is carried out by solid-phase extraction. In some embodiments, the desalting treatment comprises the following steps:

[0041] (1) Pretreat the C18 solid-phase extraction cartridge successively with methanol and 70% acetonitrile (containing 0.1% formic acid);

[0042] (2) Elute the sample after enzymatic digestion on the pretreated C18 solid-phase extraction cartridge with 0.1% formic acid and 70% acetonitrile (containing 0.1% formic acid);

[0043] (3) Dry the eluate and dissolve it with 0.1% formic acid for mass spectrometry detection.

[0044] According to another aspect of the present disclosure, there is provided a kit for quantitative detection of Amuc_1100 protein for a sample to be tested, characterized in that the kit comprises a polypeptide standard, the polypeptide standard comprising 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, 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 as 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, there is provided an application of the kit described in the present disclosure in the identification and / or quantitative detection of Amuc_1100 protein. The kit detects polypeptides of Amuc_1100 protein in a sample to be tested by liquid chromatography tandem mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Shows the mass spectrometry precursor ion results and secondary spectra of 4 Amuc_1100 protein characteristic peptides in some embodiments according to the present disclosure.

[0051] Figure 2 Shows the mass spectrometry precursor ion results and secondary spectra of 4 Amuc_1100 characteristic peptides for the identification of Akkermansia muciniphila in some embodiments according to the present disclosure.

[0052] Figure 3 Shows the standard curve of the characteristic peptides in some embodiments according to the present disclosure.

[0053] Figure 4 Shows the chromatogram and mass spectrometry of the characteristic peptide AINSLVNK in different samples.

[0054] Figure 5 Shows the technical roadmap of the present application.

[0055] DEPOSIT OF STRAINS

[0056] The strain Akkermansia muciniphila ( Akkermansia muciniphila ) MNH19250 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with the deposit number GDMCC No: 63782, the deposit date being June 21, 2024, and the address being the 5th floor of Building 59, 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 .

[0057] The strain Lactobacillus crispatus Lactobacillus crispatus MNH22076 was deposited at the Guangdong Provincial Microbial Culture Collection Center, with the deposit number GDMCC No∶65554, the deposit date being November 28, 2024, and the address being the 5th floor of Building 59, 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 OF THE EMBODIMENTS

[0058] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0059] The reagents and / or reagent kits used in the following embodiments were all commercially obtained or could be synthesized by known methods.

[0060] The instruments and reagents involved in the following embodiments are shown in Tables 1 and 2 below:

[0061]

[0062]

[0063] Example 1: Determination of characteristic peptides for Amuc_1100 protein identification

[0064] In this example, using the Amuc_1100 recombinant protein as a standard sample, the protein was enzymatically digested into polypeptides, and full scanning was performed in the data-dependent full scan (Full MS / data dependent MS2) mode on the UPLC-QE-MS platform. Analysis was carried out using Proteome Discoverer 2.5 software to screen and determine the characteristic peptides of the Amuc_1100 protein.

[0065] 1. Sample

[0066] The Amuc_1100 recombinant protein, derived from Escherichia coli, was purchased from Wuhan Fine Biotech Co., Ltd.

[0067] 2. Experimental procedure

[0068] 2.1 Protease digestion

[0069] For the Amuc_1100 recombinant protein standard, take 50 μg and dissolve it in 8 M urea (24.0240 g of urea and 0.6057 g of Tris base are dissolved in 40 mL of pure water, the pH is adjusted to 8.0 with hydrochloric acid, and the volume is made up to 50 mL with pure water). Add DTT (100 mM) to the protein solution to make its final concentration 10 mmol / L, reduce at 37°C for 60 min, then add IAA (100 mM) to make its final concentration 50 mM, incubate in the dark at room temperature for 45 min. Finally, dilute the urea concentration of the sample to less than 1 M, add trypsin at a mass ratio of 1:50 (trypsin:protein), and digest overnight at 37°C.

[0070] 2.2 Desalting treatment of peptides

[0071] Install the Waters SEP-PAK C18 solid-phase extraction cartridge into a 15 mL centrifuge tube, add 1 mL of methanol and centrifuge at 150 r / min for 1 min, then add 0.5 mL of 70% acetonitrile (containing 0.1% formic acid) and centrifuge at 150 r / min for 1 min; load the digested sample, centrifuge at 150 r / min for 3 min, add 1 mL of 0.1% formic acid, and centrifuge at 150 r / min for 3 min, repeat 3 times; take out the solid-phase extraction cartridge, install it into a new 15 mL centrifuge tube, add 0.5 mL of 70% acetonitrile (containing 0.1% formic acid), and centrifuge at 150 r / min for 3 min, repeat once; the eluate is the desalted peptides. Dry the eluate, dissolve it in 100 μL of 0.1% formic acid, centrifuge at 10000 r / min for 10 min, take the supernatant and freeze-dry it for subsequent mass spectrometry detection.

[0072] 2.3 Mass spectrometry detection of characteristic peptides

[0073] The freeze-dried sample is dissolved in 0.1% formic acid aqueous solution and separated by a UPLC ultra-high performance liquid system. The peptides are ionized after separation by the ultra-high performance liquid system and then injected into an ESI ion source for ionization, and then analyzed by a Q-Exactive HF mass spectrometer. Both the peptide parent ions and their secondary fragments are detected using the high-resolution Orbitrap in the Q-Exactive HF. The conditions and parameters for ultra-high performance liquid separation and mass spectrometry detection are shown in Table 3 below.

[0074]

[0075] The mass spectrometry data was processed using Proteome Discoverer 2.5 software to identify and screen for characteristic peptides. Peptides containing cleavage sites, missed cleavage sites, as well as those with methionine (M, prone to oxidation), glycosylation motifs of NXS / T, and peptide sequences prone to cyclization and oxidation were excluded to obtain the characteristic peptides of the target protein, as shown in Table 4.

[0076]

[0077] The detection results are shown in Table 4. By digesting the Amuc_1100 protein standard and performing mass spectrometry detection, 4 highly sensitive characteristic peptides of the Amuc_1100 protein were identified. The corresponding precursor ion results and MS / MS spectra of the peptides are shown in Figure 1 , where 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)].

[0078] Example 2: Identification and verification of Amuc_1100 characteristic peptides in Akkermansia muciniphila

[0079] 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 detection to verify the specificity of the 4 characteristic peptides identified in Example 1.

[0080] 1. Sample

[0081] The strain used in this experiment was Akkermansia muciniphila MNH19250, which was deposited in the Guangdong Provincial Culture Collection Center of Microorganisms, and the deposit name was Akkermansia muciniphila MNH19250, and the deposit number of the strain was GDMCC No: 63782.

[0082] 2. Experimental procedure

[0083] 2.1 Cultivation

[0084] The MNH19250 seed was inoculated into the activation medium (Medium 1, the formula is shown in Table 5) for activation, anaerobically cultured at 37°C for 24 - 36 h (standard OD600 ≥ 1.6), and then passaged at a ratio of 2.5% into Medium 2 (the formula is shown in Table 6), anaerobically cultured at 37°C for 48 h, and then the bacterial suspension was collected for subsequent experiments.

[0085]

[0086]

[0087] 2.2 Detection of characteristic peptides

[0088] 2.2.1 Protein extraction and digestion

[0089] Take the Akkermansia muciniphila bacterial suspension (3.5 - 4 ml), centrifuge, discard the supernatant, add 8 M urea to the precipitate, sonicate, extract, centrifuge at 12,000 r / min at low temperature for 20 min, take the supernatant, and precipitate with acetone; wash the obtained protein precipitate three times with acetone, air-dry naturally, and then redissolve it with 8 M urea solution. Add DTT to make its final concentration 10 mM, reduce at 37°C for 60 min, then add IAA to make its final concentration 50 mM, incubate at room temperature in the dark for 45 min, and finally dilute the urea concentration of the sample to less than 1 M, and add trypsin at a mass ratio of 1:50 (trypsin: protein precipitate), and digest overnight at 37°C.

[0090] 2.2.2 Desalting treatment of peptides

[0091] The method of this step is the same as the desalting treatment of peptides in Example 1.

[0092] 2.2.3 Mass spectrometry detection of characteristic peptides

[0093] The mass spectrometry detection method is the same as the mass spectrometry method in Example 1. The detection results are shown in Table 7:

[0094]

[0095] The 4 Amuc_1100 characteristic peptides determined in Example 1: SANAAEITPSR, ISIAAK, AINSLVNK, EQVFVQVSLNLVHFNQPK can be identified in MNH19250 bacteria. As Figure 2 , the corresponding precursor ion results and MS / MS spectra of the peptides, where; 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)].

[0096] Example 3: Method for quantitatively detecting Amuc_1100 protein by liquid chromatography tandem mass spectrometry

[0097] The principle of quantitative detection of Amuc_1100 protein is as follows: First, according to the 4 characteristic peptides of Amuc_1100 identified in Example 1, further screening is carried out based on principles such as peptide segment stability and high mass spectrometry detection sensitivity. Then, according to the mass spectrometry screening results and software simulation, the characteristic peptide AINSLVNK is preferably selected as the characteristic peptide for quantitative detection of Amuc_1100 protein. A standard curve is established with a polypeptide standard to quantify the content of Amuc_1100 protein in AKK bacteria or real samples.

[0098] 1. Sample

[0099] AINSLVNK polypeptide standard, purity 98%, molecular weight 858.50434. Synthesized by GL Biochem (Shanghai) Ltd.

[0100] 2. Experimental procedures

[0101] 2.1 Reagent preparation

[0102] 1) Stock solution of polypeptide standard

[0103] Precisely weigh 5.00 mg of the synthesized polypeptide, place it in a 5 mL volumetric flask, add 0.1% formic acid aqueous solution to dissolve and make up to the mark, shake well, and prepare a stock solution with a concentration of 1000 μg / mL.

[0104] 2) Linear solution

[0105] Precisely measure 0.1 mL of the stock solution of polypeptide standard, place it in a 10 mL volumetric flask, add 0.1% formic acid water to dilute and make up to the mark, shake well, to obtain a standard working solution of 10 mg / L. Then dilute it 10 times to get 1 mg / L. Take the prepared 1 mg / L standard working solution and sequentially dilute it to obtain standard working solutions of 500, 100, 50, 10, 5, 1 μg / L as the linear working dilution solution of the polypeptide standard.

[0106] 2.2 Standard curve drawing

[0107] Using the above-prepared linear working dilution solution of the polypeptide standard (500, 100, 50, 10, 5, 1 μg / L), the content of the characteristic peptide in the linear working dilution solution of the polypeptide standard is detected by a triple quadrupole liquid chromatography-mass spectrometry instrument, and a standard curve is drawn for subsequent quantification of the content of the characteristic peptide in real samples using the standard curve, and finally the content of Amuc_1100 protein is calculated.

[0108] The detection conditions of high performance liquid chromatography-tandem mass spectrometry are shown in Table 8 below.

[0109]

[0110] The detection results of the standard curve are shown in Table 9 and Figure 3As shown, the results show that the standard product for detecting the concentration of characteristic peptides by mass spectrometry is used, and a linear equation is fitted according to the peptide concentration (ng / ml) and the response of the peptides 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 the peptide concentration, and the deviation between the concentration detected by mass spectrometry and the standard concentration is about 10%. Therefore, this standard curve can be used to quantify this characteristic peptide in real samples, and finally the content of Amuc_1100 protein in the samples can be obtained through conversion.

[0111]

[0112] Example 4: Quantitative detection of Amuc_1100 protein in Akkermansia muciniphila

[0113] To evaluate the ability of the established method for quantitative detection of Amuc_1100 protein in Example 3 to detect the content of Amuc_1100 in real samples. In this example, two different forms of Akkermansia muciniphila samples were prepared, including the conventionally cultured Akkermansia muciniphila suspension sample and the Akkermansia muciniphila sample cultured with Garcinia cambogia extract (purchased from Changsha Huirui Biotechnology Co., Ltd.) (due to the insolubility of Garcinia cambogia in the culture medium, increasing the complexity of the bacterial sludge sample). In addition, Lactobacillus crispatus Lactobacillus crispatus MNH22076 was used as a negative control, and the established Amuc_1100 quantification method was used to detect the content of the actual samples.

[0114] 1. Samples

[0115] 1) Strains

[0116] Akkermansia muciniphila was MNH19250 in Example 2; Lactobacillus crispatus was Lactobacillus crispatus MNH22076, which was deposited in the Guangdong Provincial Microbial Culture Collection Center, and the deposit number of the strain was GDMCC No∶65554.

[0117] 2) Preparation of bacterial detection samples

[0118] MNH19250 conventionally cultured bacterial suspension sample (MNH19250 bacterial solution): Prepared according to the conditions in Example 2. After seed activation, it was inoculated into 100 mL of culture medium and cultured under anaerobic conditions at 37 °C for 48 h. OD600 = 2.03, and the bacterial suspension was collected;

[0119] MNH19250 fermented culture sludge sample (MNH19250 sludge): Based on medium 2, add Garcinia cambogia extract (3 g / L) to prepare the fermentation medium. After seed activation, inoculate it into 1 L of fermentation culture and culture it under anaerobic conditions at 37°C for 48 h. OD600 = 2.26, then centrifuge to remove the supernatant and collect the sludge precipitate.

[0120] MNH22076 bacterial suspension sample (MNH22076 bacterial solution): Carry out according to the conditions of Example 2. After seed activation, inoculate it into 100 mL of medium and culture it under anaerobic conditions at 37°C for 14 h. OD600 = 2.21, and collect the bacterial suspension.

[0121] 2. Experimental procedures

[0122] 2.1 Protein extraction

[0123] 1) Bacterial suspension sample: Take an appropriate amount of the sample (3.5 - 4 ml), centrifuge, discard the supernatant, add 8 M urea to the precipitate for ultrasonic disruption, extraction, centrifuge at 12,000 r / min at low temperature for 20 min, take the supernatant, and make up the volume of the supernatant to 500 μL. Take a small amount of the supernatant and use the Bradford method (Coomassie brilliant blue method) to detect the protein concentration. Add acetone to precipitate the remaining supernatant; wash the obtained protein precipitate three times with acetone, air-dry naturally, then redissolve it with 8 M urea solution and add DTT to make its final concentration 10 mM, reduce at 37°C for 60 min, then add IAA to make its final concentration 50 mM, incubate at room temperature in the dark for 45 min, and finally dilute the urea concentration of the sample to less than 1 M, and add trypsin at a mass ratio of 1:50 (trypsin:protein) and digest overnight at 37°C.

[0124] 2) Sludge sample: Add 8 M urea to an appropriate amount of the sample (about 0.5 g) for ultrasonic disruption, extraction, centrifuge at 12,000 r / min at low temperature for 20 min, take the supernatant, and make up the volume of the supernatant to 500 μL. Take a small amount of the supernatant for protein concentration detection. Add acetone to precipitate the remaining supernatant; wash the obtained protein precipitate three times with acetone, air-dry naturally, then redissolve it with 8 M urea solution. Add DTT to the protein solution to make its final concentration 10 mM, reduce at 37°C for 60 min, then add IAA to make its final concentration 50 mM, incubate at room temperature in the dark for 45 min, and finally dilute the urea concentration of the sample to less than 1 M, and add trypsin at a mass ratio of 1:50 (trypsin:protein) and digest overnight at 37°C.

[0125] 3) Detection of total protein content in the sample (Bradford method)

[0126] (1) Preparation of Bradford concentrated staining solution: 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 staining solution can be stored stably at 4 °C for at least 6 months.

[0127] (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 to make a standard curve.

[0128] (3) Dissolve the sample to be tested in a buffer solution, which should be the same as the buffer solution used for making the standard curve (preferably PBS).

[0129] (4) Dilute the concentrated dye-binding solution with distilled water at a ratio of 1:4. If precipitation occurs, filter it off.

[0130] (5) Add 5 ml of the diluted dye-binding solution to each sample and incubate for 5 - 30 min. After the dye binds to the protein, the solution will change from red to blue. Measure the absorbance at a wavelength of 595 nm. Note that the color development reaction should not exceed 30 min.

[0131] (6) Calculate the protein concentration of the sample to be tested according to the standard curve, and finally calculate the total protein content based on the protein concentration and the volume of the sample protein extraction solution.

[0132] 2.2 Desalting treatment of peptide segments

[0133] The desalting method of peptide segments is the same as that in Example 1.

[0134] 2.3 Quantitative detection of Amuc_1100 protein in samples

[0135] Refer to the method in Example 3, use the characteristic peptide standard to draw a standard curve, and then detect the content of the characteristic peptide in the sample by a triple quadrupole liquid chromatography - mass spectrometry. Figure 4 The chromatograms of different samples and the mass spectrum of the characteristic peptide AINSLVNK are shown respectively.

[0136] 2.4 Calculation method for the content of test samples

[0137] 1) The calculation formula can be used to convert the content of Amuc_1100 in liquid samples or solid samples. The formula is as follows:

[0138] Content of Amuc_1100 in the test sample (ng / mg or ng / mL) = (CX - C0) × M1 × V / m / M2 / 1000 (Formula 1)

[0139] m: volume of liquid (volume of the sample, in liters (L)) or mass of solid (mass of the sample, in grams (g));

[0140] M1: molecular mass of protein (molecular weight of the target protein), the Amuc_1100 protein is 34213 Da;

[0141] M2: molecular mass of polypeptide standard (molecular weight of the characteristic peptide), the polypeptide is 858.50434 Da;

[0142] V: final volume of the sample solution after volume fixation, in milliliters (mL), referring to the final volume of the dissolved desalted and freeze-dried peptide segments, i.e., 0.1 mL;

[0143] C0: mass concentration of the blank, in micrograms per liter (μg / L);

[0144] CX: mass concentration of the polypeptide in the sample to be tested, in micrograms per liter (μg / L).

[0145] 2) Content of Amuc_1100 protein per milligram of total bacterial protein (ng / mg) = Content of Amuc_1100 in the sample / Amount of total bacterial protein (Formula 2)

[0146] 2.5 Detection Results and Analysis of Amuc_1100 in Samples

[0147] Through the quantitative detection of the concentration of the protein extraction solution for three samples of MNH19250 bacterial liquid, MNH19250 bacterial sludge, and MNH22076 bacterial liquid, the contents of total bacterial protein in the samples were finally determined to be 0.43 mg, 1.30 mg, and 0.71 mg respectively. The results in Table 10 show that the content of the characteristic peptide of Amuc_1100 protein in the samples was detected by mass spectrometry, and the results were: 5.68 ng / mL, 21.18 ng / mL, and 0 ng / mL. Finally, through the calculation of Formula 1, the content of Amuc_1100 protein in each sample was: 5.66 ng / mL, 0.16 ng / mg, and 0 ng / mg (i.e., not detected). In addition, the content of Amuc_1100 protein per milligram of total bacterial protein was calculated by Formula 2, and it was found that in the AKK bacterial liquid sample, the content of Amuc_1100 protein per milligram of total bacterial protein was 52.65 ng; in the AKK bacterial sludge sample, the content of Amuc_1100 protein per milligram of total bacterial protein was 64.6 ng. The results indicate that the established quantitative detection method could not detect Amuc_1100 in the negative control strain Lactobacillus crispatus, while the content of Amuc_1100 protein could be quantified in Akkermansia muciniphila obtained by two different preparation methods, and the contents were similar, verifying the accuracy of the detection method.

[0148]

[0149] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A method for quantitatively detecting Amuc_1100 protein, characterized in that, The method comprises detecting a polypeptide of the Amuc_1100 protein in a sample to be tested by liquid chromatography tandem mass spectrometry, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1 or SEQ ID NO: 2; The sample to be tested is a bacterial liquid or bacterial sludge containing Akkermansia muciniphila; The quantitative detection comprises the steps of synthesizing a standard of the polypeptide and obtaining a standard curve of the polypeptide according to the mass spectrometry signal intensity of the synthesized standard; 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; The chromatographic column is a C18 column; Detection was performed using a triple quadrupole liquid spectrometer.

2. The method according to claim 1, 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) Quantitatively analyze the Amuc_1100 protein in the sample to be tested based on the obtained mass spectrometry data.

3. The method according to claim 2, 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; In the enzymatic hydrolysis treatment, the mass ratio of trypsin to the sample containing the Amuc_1100 protein is 1:(20-100); the temperature of the enzymatic hydrolysis treatment is 35-40° C.; and the time of the enzymatic hydrolysis treatment is 4-24 hours.

4. The method according to claim 1, wherein 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; 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: 2 in the mass spectrometer is 601-604.

5. Use of a polypeptide in quantitatively detecting Amuc_1100 protein, characterized in that, The quantitative detection is performed by the method according to any one of claims 1 to 4, wherein the polypeptide of the Amuc_1100 protein in the test sample is detected by liquid chromatography tandem mass spectrometry, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1 or SEQ ID NO: 2; The sample to be tested is a bacterial liquid or bacterial sludge containing Akkermansia muciniphila; 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 application according to claim 5, wherein 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; 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: 2 in the mass spectrometer is 601-604.

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