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 invention belongs to the field of biological detection, and in particular relates to a method and a kit for quantifying Amuc_1100 protein. Background Art

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

[0003] As research deepens, the study of Akk bacteria and diseases has shifted from correlation issues to clear causal relationships and analysis of mechanistic effects. Not only live Akk bacteria, but also pasteurized Akk and Akk bacteria components, such as outer membrane proteins, secretory proteins, and exosomes, have been shown to play a mechanistic role in disease regulation (PMID: 36835309). Amuc_1100 is a specific protein of the outer membrane of Akk bacteria. As one of the specific and unique molecular mechanisms of Akk bacteria acting on host health, recent studies have found that Amuc_1100 can reproduce the beneficial effects of Akk bacteria on diseases (metabolic diseases, inflammation, and tumors, etc.). For example, Amuc_1100 improves the metabolism of obese and diabetic mice through the TLR2 pathway (PMID: 27892954); in the mouse colitis model, Amuc_1100 can improve colitis by reducing the macrophages and cytotoxic T lymphocytes (CTLs) infiltrating the colon, and in the mouse colon cancer model, Amuc_1100 can inhibit ulcerative colitis-related cancer by inducing TNF-α and inhibiting PD-1 expression, amplification and activation of CTL (PMID: 32169907); Amuc_1100 can alleviate the depressive-like behavior of chronic unpredictable mild stress (CUMS)-induced depressed mice, and improve the downregulation of brain-derived neurotrophic factor (BDNF) and inflammatory response in the hippocampus (PMID: 34129964). These evidences indicate that Amuc_1100 protein is an important active substance and mechanism effector of Akk bacteria in exerting probiotic effects on diseases.

[0004] Based on the above considerations about the role of Akk bacteria in diseases and the effect of its active substance Amuc_1100 on the potential efficacy of Akk strains in diseases, Amuc_1100 can be used as one of the potential evaluation active markers of Akk bacteria's potential activity and function, or Akk product efficacy, or clinically related disease indicators, which is of great significance for the future development, application and commercialization of Akk bacteria in the fields of food and medicine. Therefore, the identification and quantitative detection of Amuc_1100 protein is an important prerequisite for the functional evaluation of Akk strains or their future application in different scenarios.

[0005] For protein quantitative detection, it can be mainly achieved by antibody or mass spectrometry. At present, the detection of Amuc_1100 protein content in Akk strains has only been reported to be achieved by antibody methods, but there are no commercialized antibodies or detection kits for Amuc_1100 protein on the market. Most of the antibody methods reported at home and abroad are prepared by scientific research institutions themselves by immunizing animals with Amuc_1100 recombinant protein (PMID: 27892954). Animal immunization experiments are greatly affected by animal immune responses, and the antibody content obtained is low. For large-scale antibody extraction, the method needs to be optimized. In addition, the production of Amuc_1100 antibodies also involves a long time and cost. These reasons have temporarily greatly limited the detection and application of Amuc_1100 protein. Therefore, it is necessary to develop new methods for the identification and quantification of Amuc_1100 protein. So far, there has been no literature report on mass spectrometry methods for protein identification and quantitative detection of Akk strains or samples containing Akk strains using Amuc_1100 protein characteristic peptides. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present disclosure provides a method for accurately, stably and quickly quantitatively detecting Amuc_1100 protein. The present disclosure has discovered a group of polypeptides that can be used to identify or quantitatively detect Amuc_1100 protein, and such polypeptides can achieve stable detection of Amuc_1100 protein. Further, a polypeptide standard is synthesized from a standard protein to establish a standard curve for stable, accurate and quantitative detection of the content of Amuc_1100 protein in a sample to be tested.

[0007] According to one aspect of the present disclosure, there is provided an application of a polypeptide in the quantitative detection of Amuc_1100 protein, wherein the quantitative detection detects the polypeptide in the Amuc_1100 protein in the test sample 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 the characteristic peptides prepared by the standard protein are plotted into a standard curve, so as to facilitate stable and accurate quantitative detection of Amuc-1100 protein in the sample.

[0009] In some embodiments, the quantitative detection includes 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.

[0010] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide with an amino acid sequence as shown in SEQ ID NO: 3 in the mass spectrometer 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 polypeptide with an amino acid sequence as shown in SEQ ID NO: 3 in the mass spectrometer 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 polypeptide with the amino acid sequence as shown in SEQ ID NO: 1 in the mass spectrometer 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 polypeptide with the amino acid sequence as shown in SEQ ID NO: 1 in the mass spectrometer 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 polypeptide with an amino acid sequence as shown in SEQ ID NO: 2 in the mass spectrometer 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 polypeptide with an amino acid sequence as shown in SEQ ID NO: 2 in the mass spectrometer 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 by the polypeptide with an amino acid sequence as shown in SEQ ID NO: 4 in the mass spectrometer is 557-560, for example, 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 an amino acid sequence as shown in SEQ ID NO: 4 in the mass spectrometer is 558-559, for example, 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 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 includes 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.

[0017] In some embodiments, the mass-to-charge ratio of the parent ion of the detection signal generated by the polypeptide with an amino acid sequence as shown in SEQ ID NO: 3 in the mass spectrometer 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 polypeptide with an amino acid sequence as shown in SEQ ID NO: 3 in the mass spectrometer 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 polypeptide with the amino acid sequence as shown in SEQ ID NO: 1 in the mass spectrometer 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 polypeptide with the amino acid sequence as shown in SEQ ID NO: 1 in the mass spectrometer 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 polypeptide with an amino acid sequence as shown in SEQ ID NO: 2 in the mass spectrometer 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 polypeptide with an amino acid sequence as shown in SEQ ID NO: 2 in the mass spectrometer 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 by the polypeptide with an amino acid sequence as shown in SEQ ID NO: 4 in the mass spectrometer is 557-560, for example, 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 an amino acid sequence as shown in SEQ ID NO: 4 in the mass spectrometer is 558-559, for example, 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, 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 used for elution, 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 spectrometer is used for detection.

[0026] In some embodiments, the method comprises the steps of: (1) using trypsin to enzymatically hydrolyze the sample containing Amuc_1100 protein, and then desalt it; (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 sample to be tested based on the obtained mass spectrometry data.

[0027] 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 is extracted, followed by enzymatic hydrolysis and desalting treatment.

[0028] In some embodiments, in the enzymatic hydrolysis treatment, the mass ratio of trypsin to the sample 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.

[0029] 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.

[0030] 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.

[0031] In some embodiments, the enzymatic treatment is performed using methods well known in the art.

[0032] In some embodiments, the enzymatic treatment comprises the following steps: (1) Dissolve the sample in a urea-Tris solution (final concentration 5-10 M) to obtain a protein solution; (2) adding DTT solution (final concentration of 8-15 mM) to the protein solution for reduction treatment (35-40°C, 30-120 min), and then adding IAA solution (final concentration of 20-100 mM) for incubation (room temperature, protected from light, 20-90 min); (3) After diluting the urea concentration in the sample to be tested to less than 1 M, add trypsin for enzymatic hydrolysis.

[0033] In some embodiments, the desalting treatment is performed using methods well known in the art.

[0034] In some embodiments, the desalting process is carried out by solid phase extraction. In some embodiments, the desalting process comprises the following steps: (1) The C18 solid phase extraction column was pretreated with methanol and 70% acetonitrile (containing 0.1% formic acid) in sequence; (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); (3) The eluate was dried and dissolved in 0.1% formic acid for mass spectrometry detection.

[0035] According to another aspect of the present disclosure, a kit for quantitative detection of Amuc_1100 protein in a sample to be tested is provided, characterized in that the kit comprises a polypeptide standard, the polypeptide standard comprises at least one polypeptide having an amino acid sequence of SEQID 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.

[0036] In some embodiments, the amino acid sequence of the polypeptide standard is as shown in SEQ ID NO: 3.

[0037] In some embodiments, the kit further comprises trypsin.

[0038] In some embodiments, the kit further comprises at least one of a urea solution, a DTT solution, and an IAA solution.

[0039] In some embodiments, the kit further comprises at least one of acetone, methanol, and acetonitrile.

[0040] According to another aspect of the present disclosure, provided is a use of the kit described in the present disclosure in identification and / or quantitative detection of Amuc_1100 protein, wherein the kit detects the polypeptide of Amuc_1100 protein in a sample to be tested by liquid chromatography tandem mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The mass spectrometry precursor ion results and secondary spectra of four Amuc_1100 protein characteristic peptides according to some embodiments of the present disclosure are shown.

[0042] 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.

[0043] Figure 3 A standard curve of characteristic peptides according to some embodiments of the present disclosure is shown.

[0044] Figure 4 Chromatograms and mass spectra of the characteristic peptide AINSLVNK in different samples are shown.

[0045] Figure 5 The technical roadmap of this application is shown.

[0046] Deposit of strains The strain Akkermansia muciniphila ( Akkermansia muciniphila )MNH19250, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 63782, the deposit date is 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, the proposed taxonomic name is Akkermansia muciniphila .

[0047] Lactobacillus crispatus Lactobacillus crispatus MNH22076 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No∶65554 and the deposit date is November 28, 2024. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the deposit name is Lactobacillus crispatus MNH22076, the proposed taxonomic name is Lactobacillus crispatus . DETAILED DESCRIPTION

[0048] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0049] The reagents and / or kits used in the following examples are all commercially available or can be synthesized by known methods.

[0050] The instruments and reagents involved in the following examples are shown in Tables 1 and 2:

[0051]

[0052] Example 1: Characteristic peptide determination of Amuc_1100 protein In this example, Amuc_1100 recombinant protein was used as a standard sample, the protein was hydrolyzed into peptides by protease, and full scan was performed under the data dependent full scan (Full MS / data dependent MS2) mode of the UPLC-QE-MS platform. The analysis was performed using Proteome Discoverer 2.5 software to screen and determine the characteristic peptides of the Amuc_1100 protein.

[0053] 1. Sample Amuc_1100 recombinant protein, derived from Escherichia coli, was purchased from Wuhan Fine Biotechnology Co., Ltd.

[0054] 2. Experimental Procedure 2.1 Protease cleavage Take 50 μg of Amuc_1100 recombinant protein standard and dissolve it in 8 M urea (24.0240 g urea, 0.6057 g Tris base dissolved in 40 mL pure water, adjusted to pH = 8.0 with hydrochloric acid, and 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 it at 37°C for 60 min, then add IAA (100 mM) to make its final concentration 50 mM, incubate it at room temperature in the dark for 45 min, and 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 enzymatically hydrolyze it at 37°C overnight.

[0055] 2.2 Peptide desalting The Waters SEP-PAK C18 solid phase extraction column was placed in a 15 mL centrifuge tube, 1 mL of methanol was added and centrifuged at 150 r / min for 1 min, then 0.5 mL of 70% acetonitrile (containing 0.1% formic acid) was added and centrifuged at 150 r / min for 1 min; the enzymatically hydrolyzed sample was loaded and centrifuged at 150 r / min for 3 min, 1 mL of 0.1% formic acid was added and centrifuged at 150 r / min for 3 min, and this was repeated 3 times; the solid phase extraction column 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 centrifuged at 150 r / min for 3 min, and this was repeated once; the eluent was the desalted peptide segment, the eluent was blown dry, dissolved with 100 μL of 0.1% formic acid, centrifuged at 10000 r / min for 10 min, the supernatant was lyophilized, and then subjected to mass spectrometry detection.

[0056] 2.3 Mass spectrometry detection of characteristic peptides The lyophilized samples were dissolved in 0.1% formic acid aqueous solution and separated by UPLC ultra-high performance liquid phase system. After being separated by ultra-high performance liquid phase system, the peptides were injected into the ESI ion source for ionization and then analyzed by Q-Exactive HF mass spectrometry. The peptide parent ions and their secondary fragments were detected by high-resolution Orbitrap in Q-Exactive HF. The conditions and parameters of ultra-high performance liquid phase separation and mass spectrometry detection are shown in Table 3 below.

[0057]

[0058] The mass spectrometry data were analyzed using Proteome Discoverer 2.5 software to identify and screen the characteristic peptides, excluding those containing mis-cleavage or missed cleavage sites, glycosylation motifs containing methionine (M, easily oxidized), NXS / T, and peptide sequences that are easily cyclized and easily oxidized, to obtain the characteristic peptides of the target protein, as shown in Table 4.

[0059]

[0060] The test results are shown in Table 4. By performing enzymatic hydrolysis and mass spectrometry on the Amuc_1100 protein standard, four highly sensitive Amuc_1100 protein characteristic peptides 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)].

[0061] Example 2: Identification and verification of the Amuc_1100 characteristic peptide in Akkermansia muciniphila 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.

[0062] 1. Sample The strain used in this experiment was Akkermansia muciniphila MNH19250, which was deposited in Guangdong Microbiological Culture Collection Center under the name of Akkermansia muciniphila MNH19250, the deposit number of the strain is GDMCC No: 63782.

[0063] 2. Experimental Procedure 2.1 Training MNH19250 seeds were inoculated in 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 in medium 2 (the formula is shown in Table 6) at a ratio of 2.5%, cultured anaerobically at 37°C for 48 h, and then the bacterial suspension was collected for subsequent experiments.

[0064]

[0065]

[0066] 2.2 Characteristic peptide detection 2.2.1 Protein extraction and enzyme digestion Take the Akkermansia muciniphila suspension (3.5-4 ml), centrifuge, discard the supernatant, add 8M urea to the precipitate, ultrasonically break it, extract it, centrifuge it at 12000r / min for 20 minutes, take the supernatant, add acetone to precipitate it; wash the obtained protein precipitate with acetone three times, dry it naturally, and then re-dissolve it with 8M urea solution. Add DTT to make the final concentration of 10 mM, reduce it at 37°C for 60 minutes, then add IAA to make the final concentration of 50 mM, incubate it at room temperature in the dark for 45 minutes, and finally dilute the urea concentration of the sample to less than 1 M, add trypsin at a mass ratio of 1:50 (trypsin: protein precipitate), and enzymatically digest it at 37°C overnight.

[0067] 2.2.2 Peptide desalting The method of this step is the same as the peptide desalting treatment in Example 1.

[0068] 2.2.3 Mass spectrometry detection of characteristic peptides The mass spectrometry detection method is the same as that in Example 1. The detection results are shown in Table 7:

[0069] The four characteristic peptides of Amuc_1100 determined in Example 1: SANAAEITPSR, ISIAAK, AINSLVNK, EQVFVQVSLNLVHFNQPK can be identified in MNH19250. Figure 2 , the corresponding parent 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)].

[0070] Example 3: Method for quantitative detection of Amuc_1100 protein using liquid chromatography tandem mass spectrometry The principle of quantitative detection of Amuc_1100 protein is as follows: first, according to the 4 Amuc_1100 characteristic peptides identified in Example 1, further screening is carried out based on the principles of peptide stability and high sensitivity of mass spectrometry detection, and then, according to 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, and a standard curve is established by using polypeptide standards to quantify the content of Amuc_1100 protein in AKK bacteria or real samples.

[0071] 1. Sample AINSLVNK peptide standard, purity 98%, molecular weight 858.50434. Synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0072] 2. Experimental Procedure 2.1 Reagent Configuration 1) Peptide standard stock solution Accurately weigh 5.00 mg of synthetic peptide and place it in a 5 mL volumetric flask. Add 0.1% formic acid aqueous solution to dissolve and dilute to the mark. Shake well to prepare a stock solution with a concentration of 1000 μg / mL.

[0073] 2) Linear solution Accurately measure 0.1 mL of peptide standard stock solution, place in a 10 mL volumetric flask, add 0.1% formic acid water to dilute and dilute to the mark, shake well to obtain a 10 mg / L standard working solution, and dilute 10 times to obtain 1 mg / L. Take the prepared 1 mg / L standard working solution and dilute it in sequence to obtain 500, 100, 50, 10, 5, and 1 μg / L standard working solutions as the peptide standard linear working dilutions.

[0074] 2.2 Standard curve drawing 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 so that the standard curve could be used to quantify the characteristic peptide content in the real sample and finally convert the Amuc_1100 protein content.

[0075] The HPLC-MS / MS detection conditions are shown in Table 8 below.

[0076]

[0077] The standard curve test results are shown in Table 9 and Figure 3 As shown in the figure, the results show that the mass spectrometer detects the standard of characteristic peptide concentration, and fits the linear equation according to the peptide concentration (ng / ml) and the response (Responses) of the peptide detected by the mass spectrometer. The formula is: y = 424.313931 × x - 74.551784, R 2 = 0.99961056, indicating that the peptide response signal is linearly related to the peptide concentration, and the concentration detected by mass spectrometry is about 10% different from the standard concentration. Therefore, this standard curve can be used to quantify this characteristic peptide in the real sample, and finally the Amuc_1100 protein content of the sample can be obtained by conversion.

[0078]

[0079] Example 4: Quantitative detection of Amuc_1100 protein in AKK bacteria In order to evaluate the method for quantitative detection of Amuc_1100 protein established in Example 3, the ability of real samples to detect Amuc_1100 content was investigated. In this example, two different forms of Akk bacteria samples were prepared, including a conventional cultured Akk bacteria suspension sample and an Akk bacteria sample cultured with Garcinia cambogia extract (purchased from Changsha Huirui Biotechnology Co., Ltd.) (Garcinia cambogia is insoluble in the culture medium, which increases the complexity of the bacterial mud sample). In addition, Lactobacillus crispatus ( Lactobacillus crispatus MNH22076) was used as a negative control, and the established Amuc_1100 quantitative method was used to detect the content of the actual sample.

[0080] 1. Sample 1) strain Akkermansia muciniphila is MNH19250 in Example 2; Lactobacillus crispatus is Lactobacillus crispatusMNH22076 is deposited in Guangdong Microbiological Culture Collection Center, and the strain collection number is GDMCC No: 65554.

[0081] 2) Sample preparation for bacterial detection MNH19250 conventional culture bacterial suspension sample (MNH19250 bacterial suspension): according to the conditions of Example 2, the seeds were activated and inoculated into 100 mL of culture medium, cultured at 37°C under anaerobic conditions for 48 h, OD600 = 2.03, and the bacterial suspension was collected; MNH19250 fermentation culture sludge sample (MNH19250 sludge): Based on culture medium 2, add Garcinia cambogia extract (3g / L) to prepare the fermentation medium. After the seeds are activated, they are inoculated into 1L fermentation culture and cultured at 37℃ under anaerobic conditions for 48h. OD600=2.26. Then, the supernatant is removed by centrifugation and the sludge precipitate is collected.

[0082] MNH22076 bacterial suspension sample (MNH22076 bacterial liquid): According to the conditions of Example 2, the seeds were activated and inoculated into 100 mL of culture medium, cultured at 37° C. under anaerobic conditions for 14 h, OD600 = 2.21, and the bacterial suspension was collected.

[0083] 2. Experimental Procedure 2.1 Protein extraction 1) Bacterial suspension sample: Take an appropriate amount of sample (3.5-4ml), centrifuge, discard the supernatant, add 8M urea to the precipitate, ultrasonically break it, extract, centrifuge at 12000r / min for 20min, take the supernatant, and make up the supernatant volume to 500 μL, and take a small amount of supernatant for protein concentration detection using the Bradford method (Coomassie Brilliant Blue method). Add acetone to the remaining supernatant for precipitation; wash the resulting protein precipitate with acetone three times, dry it naturally, re-dissolve it with 8M urea solution, add DTT to a final concentration of 10 mM, reduce it at 37°C for 60min, then add IAA to a final concentration of 50 mM, incubate it at room temperature in the dark for 45min, and 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 enzymatically digest it at 37°C overnight.

[0084] 2) Mud sample: Take an appropriate amount of sample (about 0.5g), add 8M urea, ultrasonically break it, extract it, centrifuge it at 12000r / min for 20min, take the supernatant, and make up the supernatant volume to 500 μL, and take a small amount of supernatant for protein concentration detection. Add acetone to the remaining supernatant for precipitation; wash the resulting protein precipitate with acetone three times, dry it naturally, and then re-dissolve it with 8M urea solution. Add DTT to the protein solution to make the final concentration of 10 mM, reduce it at 37°C for 60min, then add IAA to make the final concentration of 50 mM, incubate it at room temperature in the dark for 45min, and 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 enzymatically hydrolyze it at 37°C overnight.

[0085] 3) Sample total protein content detection (Bradford method) (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.

[0086] (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.

[0087] (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).

[0088] (4) Dilute the concentrated dye-binding solution with distilled water at a ratio of 1:4. If a precipitate appears, remove it by filtration.

[0089] (5) Add 5 ml of diluted dye binding solution to each sample and allow to react for 5 to 30 minutes. After the dye binds to the protein, it will change from red to blue, and its absorbance will be measured at a wavelength of 595 nm. Note that the color reaction should not exceed 30 minutes. (6) Calculate the protein concentration of the sample to be tested based on the standard curve, and finally calculate the total protein content using the protein concentration and the volume of the sample protein extract.

[0090] 2.2 Peptide desalting The peptide desalting method is the same as that in Example 1.

[0091] 2.3 Quantitative detection of Amuc_1100 protein in samples 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 by triple quadrupole liquid chromatography-mass spectrometry. Figure 4 The chromatograms of different samples and the mass spectrum of the characteristic peptide AINSLVNK are shown in the figure.

[0092] 2.4 Calculation method of test sample content 1) The calculation formula can be used to convert the Amuc_1100 content in liquid samples or solid samples. The formula is as follows: The content of Amuc_1100 in the test sample (ng / mg or ng / mL) = (CX-C0) × M1 × V / m / M2 / 1000 (Formula 1) m: liquid (volume of sample, in liters (L)) or solid (mass of sample, in grams (g)); M1: protein molecular weight (target protein molecular weight), Amuc_1100 protein is 34213 Da; M2: molecular mass of peptide standard (characteristic peptide molecular weight), the peptide is 858.50434 Da; 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; C0: mass concentration of blank, in micrograms per liter (μg / L); CX: The mass concentration of the peptide to be tested, in micrograms per liter (μg / L).

[0093] 2) Amuc_1100 protein content per mg of total bacterial protein (ng / mg) = Amuc_1100 content of sample / total bacterial protein (Formula 2) 2.5 Sample Amuc_1100 test results and analysis The three samples of MNH19250 bacterial solution, MNH19250 bacterial mud and MNH22076 bacterial solution were quantitatively tested for the concentration of protein extracts, and the total bacterial protein content in the samples was 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, the content of Amuc_1100 protein in each sample was calculated by formula 1: 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 the Amuc_1100 protein content per milligram of total bacterial protein in the AKK bacterial liquid sample was 52.65 ng; the Amuc_1100 protein content per milligram of total bacterial protein in the AKK bacterial mud sample was 64.6 ng. The results showed that the established quantitative detection method could not detect Amuc_1100 in the negative control strain Lactobacillus crispatus, but the Amuc_1100 protein content could be quantified in Akkermansia muciniphila obtained by two different preparation methods, and the content was similar, which verified the accuracy of the detection method.

[0094]

[0095] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All 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 quantitative detection of Amuc_1100 protein, characterized in that: The quantitative detection detects the polypeptide of 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.

2. The use according to claim 1, 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.

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 as 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 as 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 as 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 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 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, wherein the gradient elution procedure 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 chromatographic column; and / or A triple quadrupole liquid spectrometer was used for detection.

7. The method according to claim 4, characterized in that The method comprises the following steps: (1) using trypsin to enzymatically hydrolyze the sample containing Amuc_1100 protein, and then desalt it; (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 sample to be tested 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, the Akkermansia muciniphila is lysed and bacterial protein is extracted, and then enzymatic hydrolysis and desalting are performed; 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 as 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 as 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 as 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 quantitative detection of Amuc-1100 protein in a sample to be tested, characterized in that: The kit includes a polypeptide standard, which includes 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 includes detecting the sample to be tested by liquid chromatography tandem mass spectrometry.

11. The kit according to claim 10, characterized in that The kit also includes trypsin.

12. Use of the kit according to claim 10 or 11 in the identification and / or quantitative detection of Amuc_1100 protein, characterized in that: The kit detects the polypeptide of Amuc_1100 protein in the sample to be tested by liquid chromatography tandem mass spectrometry.

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