CN114106101B - Characteristic peptide of mucin-Achroman and application thereof - Google Patents
Characteristic peptide of mucin-Achroman and application thereof Download PDFInfo
- Publication number
- CN114106101B CN114106101B CN202111175902.5A CN202111175902A CN114106101B CN 114106101 B CN114106101 B CN 114106101B CN 202111175902 A CN202111175902 A CN 202111175902A CN 114106101 B CN114106101 B CN 114106101B
- Authority
- CN
- China
- Prior art keywords
- sample
- mucin
- ackermans
- minutes
- liquid chromatography
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Dispersion Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a mucin-Achroman characteristic peptide and application thereof. The research of the invention discovers that the characteristic polypeptide of the mucin-Ackermans (Akkermansia muciniphila) can be used for detection and identification of the mucin-Ackermans, the amino acid sequence of the characteristic polypeptide provided by the invention is at least one of LLDEGQAGDNVGLLLR, LMPVEDVFSISGR, VDFNVPLK and TAAEYDNYR, and the characteristic polypeptide is unique to the mucin-Ackermans and can be identified by using liquid chromatography tandem mass spectrometry. The method has strong specificity and high sensitivity, and can accurately identify mucin-Ackermans.
Description
Technical Field
The invention relates to the technical field of microorganism detection, in particular to a characteristic peptide of mucin-Ackermans and application thereof.
Background
mucin-Achroman (Akkermansia muciniphila, abbreviated as AKKM 8) is a gram-negative strict anaerobic bacterium which is colonized on the mucosa of the human digestive tract and specifically degrades mucin. In 2004, derrien separated from human body feces and accounting for about 1% -5% of total bacteria in intestinal tract can stimulate thickening of mucous layer of organism to maintain health condition of organism. It has been reported that the colonization is one of the signs of the body health level in people suffering from inflammatory bowel disease, autism, obesity or congenital allergy.
The invention patent with publication number CN110227085A discloses application of Akkermansia muciniphila bacteria in preparing a medicament or health care product for preventing and treating depression. The invention carries out in vivo pharmacodynamics study on the role of Akkermansia muciniphila (abbreviated as AKKM 8) in the pathogenesis and anti-depression of depression, and the result shows that Akkermansia muciniphila abundance in intestinal tracts of depression patients is obviously reduced. Animal experiment results show that the intestinal Akkermansia muciniphila abundance of depressed mice is obviously reduced. According to the invention, akkermansia muciniphila bacteria transplantation is given to a depressed mouse model, and experiments show that Akkermansia muciniphila bacteria transplantation can improve the movement distance of the mouse, reduce the immobility time of tail suspension experiments and forced swimming experiments, remarkably relieve the depressed state of the mouse, and show good anti-depression curative effect without side effects.
Further prior art disclosures are mucin-Acremonium in the treatment or prophylaxis of diabetes (publication No. CN 105030841A), in the preparation of medicaments or health-care products for the treatment or prophylaxis of obesity (publication No. CN105106245A, CN 111938158A), in the prophylaxis and treatment of depression (publication No. CN110693917A, CN 110227085A), in the prophylaxis and treatment of metabolic diseases (publication No. CN110964650A, CN 111603488A), in the culture medium of mucin-Acremonium (publication No. CN 110079474A).
All of the above applications in human diseases, the premise is that there is a means for detecting whether mucin-Achroman is present or not and how the content varies in human digestive tract. In 2017 Zhangling et al (Zhangling, chenjie, qin Qianqian, etc. screening of intestinal bacteria Akkermansia muciniphila nucleic acid aptamer [ J ]. Modern preventive medicine, 2017 (17): 142-147.) 2 high-frequency nucleic acid sequence aptamers with higher affinity to mucin-Ackermans were obtained by a nucleic acid aptamer screening method, and can be used for identification of mucin-Ackermans. Qin Qianqian et al (Qin Qianqian, zhangling, wang Guoqing. Real-time fluorescent PCR quantitative detection of Akkermansia muciniphila method in feces. J. University of Sichuan university, medical edition, 2018 (1): 93-97.) in 2018, real-time fluorescent quantitative PCR detection of mucin-Ackermans in feces using upstream primer 5-CAGCACGTGAAGGTGGGGAC-3 and downstream primer 5-CCTTGCGGTTGGCTTCAGAT-3, but to date, identification of a chromatograph mass spectrum using a polypeptide sequence specific to mucin-Ackermans has not been reported in the literature.
Disclosure of Invention
The invention aims at solving the problems and provides a group of characteristic polypeptides for identifying mucin-Ackermans and a liquid chromatography mass spectrometry identification method of characteristic polypeptides specific to mucin-Ackermans, which have the advantages of strong uniqueness and high sensitivity and can accurately identify whether mucin-Ackermans is contained in a sample.
The group of characteristic polypeptides for identifying mucin-Ackermana provided by the invention consists of 8-16 amino acids, and has one or more of the sequence LLDEGQAGDNVGLLLR [ m/z 561.637 (z=3) or m/z841.949 (z=2) ], LMPVEDVFSISGR [ m/z 725.367 (z=2) ], VDFNNVPLK [ m/z466.261 (z=2) ] and TAAEYDNYR [ m/z 551.739 (z=2) ]; the parent mass error margin is 10.0ppm and the chip mass error margin is 0.05Da.
A characteristic peptide of mucin-akkermansia (Akkermansia muciniphila) having an amino acid sequence of at least one of LLDEGQAGDNVGLLLR, LMPVEDVFSISGR, VDFNVPLK and TAAEYDNYR.
The invention also provides application of the characteristic peptide in detection of mucin-Ackermans for non-medical diagnosis.
Preferably, the application, detection uses liquid chromatography mass spectrometry.
The invention also provides a method for detecting mucin-Ackermans for non-medical diagnosis, which uses a liquid chromatography mass spectrometry method to detect a sample, and if the characteristic peptide exists in the sample, the mucin-Ackermans exists in the sample.
Preferably, the sample to be detected is one of the following:
(1) Samples of human oral, esophageal, gastric, intestinal or pulmonary origin,
(2) Environmental samples. The environmental sample may be a soil sample, a water sample, a sludge sample, or the like.
Preferably, the method comprises the following steps: subjecting sample to be tested to enzyme digestion with protein extraction and trypsin, centrifuging and ultrafiltering the enzymolysis solution with ultrafiltration tube with molecular weight cut-off of 3000, collecting filtrate, lyophilizing, desalting, drying eluate containing polypeptide, dissolving with 0.1% formic acid, quantifying polypeptide content, introducing sample, and performing liquid chromatography C 18 And (3) carrying out spectrogram acquisition and identification by column gradient separation and high-resolution mass spectrometry.
More preferably, when liquid chromatography and mass spectrometry are combined, the sample injection amount is 0.5-1 g, and the sample injection flow rate is 10 nL/min; liquid chromatography C 18 The conditions for column gradient separation were as follows: phase A is an aqueous solution containing 0.1% formic acid and phase B is an acetonitrile solution containing 0.1% formic acid.
More preferably, the spectrogram acquisition time is 150 minutes, and the nanoliter liquid phase separation gradient is as follows:
more preferably, the parameters of the high resolution mass spectrum acquisition are as follows:
the research of the invention discovers that the characteristic polypeptide of the mucin-Ackermans (Akkermansia muciniphila) can be used for detection and identification of the mucin-Ackermans, the amino acid sequence of the characteristic polypeptide provided by the invention is at least one of LLDEGQAGDNVGLLLR, LMPVEDVFSISGR, VDFNVPLK and TAAEYDNYR, and the characteristic polypeptide is unique to the mucin-Ackermans and can be identified by using liquid chromatography tandem mass spectrometry. The method has strong specificity and high sensitivity, and can accurately identify mucin-Ackermans.
Drawings
FIG. 1 shows a parent ion mass spectrum (A) and a secondary mass spectrum (B) of polypeptide LLDEGQAGDNVGLLLR [ m/z841.949 (z=2) ].
FIG. 2 shows a parent ion mass spectrum (A) and a secondary mass spectrum (B) of polypeptide LMPVEDVFSISGR [ m/z 725.367 (z=2) ].
FIG. 3 shows a parent ion mass spectrum (A) and a secondary mass spectrum (B) of polypeptide TAAEYDNYR [ m/z 551.739 (z=2) ].
FIG. 4 shows a parent ion mass spectrum (A) and a secondary mass analysis spectrum (B) of the polypeptide VDFNNVPLK [ m/z466.261 (z=2) ].
Detailed Description
Example 1: screening for characteristic polypeptides
In the embodiment, equipment is adopted for high-resolution mass spectrum of nano liter liquid chromatograph (EASY-nLC 1000) and LTQ-Orbitrap Elite, and all reagents such as ammonia bicarbonate, trypsin, acetonitrile, formic acid and the like need to be purified by chromatography.
The fecal sample was first separated from bacteria by differential centrifugation, and then a polypeptide sample was prepared as follows: the bacterial samples were isolated by adding a 100% TCA solution at a ratio of 4:1 (V: V) followed by 1M DTT mother liquor to a final concentration of 100mM. Shaking and mixing uniformly. After 1 hour on ice, 14000g was centrifuged for 5 minutes, and the supernatant was discarded. The precipitate was added with 800. Mu.l of acetone at-20℃and the precipitate was vortex-blown off, allowed to stand at 4℃for 10 minutes, centrifuged at 14000g for 5 minutes, and the supernatant was discarded. The precipitate was added with 300. Mu.l of acetone at-20℃and the precipitate was blown off and sonicated for 15 min to aid dissolution, and allowed to stand at-20℃for 30 min. Centrifuge 6500g for 10 min at 4℃and discard the supernatant. This step was repeated 2 times. The precipitate was dried in a metal bath at 95℃for 5-10 minutes to completely remove the acetone. Fresh 8M UA (urea) solution is prepared, 120 mu l of 8M UA is added to each tube, the precipitate is blown off, the solution is placed in a metal bath at 50 ℃ for 10 minutes, 14000g is centrifuged for 10 minutes and then incubated, the supernatant is taken to obtain a bold protein solution, and the protein concentration is measured by Nano-drop. The corresponding protein solution of 300. Mu.g protein per sample was transferred to a 10K ultrafiltration tube (the tube was placed on a collection tube), 200. Mu.l 8M UA was added, 14000g was centrifuged for 10 minutes, and the waste solution in the collection tube was discarded, and this step was repeated 2 times. 100 μl of 0.05M IAA (iodoacetamide) solution was added and mixed with the liquid in the ultrafiltration tube, followed by incubation in the dark for 20 minutes; 14000g was centrifuged for 10 minutes, and the waste liquid in the collection tube was discarded. 100 μl UA was added and 14000g centrifuged for 10 minutes, the waste liquid in the collection tube was discarded, and the procedure was repeated 2 times. 100 μl of 0.05M ABC (ammonium bicarbonate) solution was added, mixed with the liquid in the ultrafiltration tube, and incubated in the dark for 20 minutes; 14000g was centrifuged for 10 minutes, and the waste liquid in the collection tube was discarded, and this step was repeated 2 times. Mu.l of a 0.05M ABC solution was added, following protein: enzyme=50-100:1, 1. Mu.g/. Mu.l trypsin stock was added, mixed and incubated at 37℃for 15h. The ultrafiltration tube was transferred to a fresh centrifuge tube and centrifuged at 14000g for 10 minutes. 40 μl of ABC solution was added and the mixture centrifuged at 14000g for 10 minutes. The ultrafiltration tube was discarded and the liquid in the centrifuge tube was lyophilized.
And (3) desalting: the above lyophilized samples were dissolved with 20 μl of 0.4% trifluoroacetic acid (TFA); wetting C with 100% acetonitrile ACN 18 Gun head (Zip Tip C) 18 Millipore) once, 10. Mu.L/time; wetting the gun head twice with 50% ACN, 10 μl/time; the Tip tips were equilibrated twice with 10 μl of 0.1% TFA; sucking 10 mu L of sample, and blowing back and forth for 10 times; wash twice with 0.1% TFA, 10 μl/time; elution was performed with 10 μl of 0.1% formic acid/60% ACN; freeze-drying for about 30 minutes; adding 20 mu L of 0.1% formic acid for dissolution, taking 1.5 mu L of sample, detecting peptide concentration by Nanodrop, transferring the residual sample into a 250 mu L upper sample tube of a mass spectrum, placing the sample into a 48-hole upper sample plate of a nano-upgrading liquid chromatograph, separating 1 mu g total amount of polypeptide by a nano-liter liquid chromatograph (EASY-nLC 1000) according to the measured polypeptide concentration, and identifying by LTQ-Orbitrap Elite by high resolution mass spectrum, wherein the mass spectrum data is analyzed and searched by PEAKS Studio 8.0, and the search parameters are as follows:
1. search engine name: PEAKS;
2. parent mass error margin: 10.0ppm;
3. fragment mass error margin: 0.05Da;
4. enzyme: trypsin;
5. fixed modification: carboxymethyl;
6. variable modification: oxidation and degradation;
7. database: derived from the bacterial protein database in Uniprot public databases.
The search results are shown in Table 1, wherein "Y" represents "Yes" and "N" represents "No" in whether or not an item is specific.
TABLE 1
The results showed that of the 9 polypeptides identified, 4 were characteristic polypeptides of mucin-Acremonium, LLDEGQAGDNVGLLLR, LMPVEDVFSISGR, VDFNVPLK and TAAEYDNYR, respectively. The remaining 5 are not, as can be retrieved in other bacteria, e.g. ehilar peptides are also present in other species, as shown in table 2, where if one term is specific "Y" means "yes" and "N" means "no".
TABLE 2
Example 2: specificity identification-positivity
In the embodiment, equipment is adopted for high-resolution mass spectrum of nano liter liquid chromatograph (EASY-nLC 1000) and LTQ-Orbitrap Elite, and all reagents such as ammonia bicarbonate, trypsin, acetonitrile, formic acid and the like need to be purified by chromatography.
A100% TCA solution was added to mucin-Achroman at a ratio of 4:1 (V: V) and 1M DTT mother liquor was added to give a final concentration of 100mM. Shaking and mixing uniformly. After 1 hour on ice, 14000g was centrifuged for 5 minutes, and the supernatant was discarded. The precipitate was added with 800. Mu.l of acetone at-20℃and the precipitate was vortex-blown off, allowed to stand at 4℃for 10 minutes, centrifuged at 14000g for 5 minutes, and the supernatant was discarded. The precipitate was added with 300. Mu.l of acetone at-20℃and the precipitate was blown off and sonicated for 15 min to aid dissolution, and allowed to stand at-20℃for 30 min. Centrifuge 6500g for 10 min at 4℃and discard the supernatant. This step was repeated 2 times. The precipitate was dried in a metal bath at 95℃for 5-10 minutes to completely remove the acetone. Fresh 8M UA (urea) solution is prepared, 120 mu l of 8M UA is added to each tube, the precipitate is blown off, the solution is placed in a metal bath at 50 ℃ for 10 minutes, 14000g is centrifuged for 10 minutes and then incubated, the supernatant is taken to obtain a bold protein solution, and the protein concentration is measured by Nano-drop. The corresponding protein solution of 300. Mu.g protein per sample was transferred to a 10K ultrafiltration tube (the tube was placed on a collection tube), 200. Mu.l 8M UA was added, 14000g was centrifuged for 10 minutes, and the waste solution in the collection tube was discarded, and this step was repeated 2 times. 100 μl of 0.05M IAA (iodoacetamide) solution was added and mixed with the liquid in the ultrafiltration tube, followed by incubation in the dark for 20 minutes; 14000g was centrifuged for 10 minutes, and the waste liquid in the collection tube was discarded. 100 μl UA was added and 14000g centrifuged for 10 minutes, the waste liquid in the collection tube was discarded, and the procedure was repeated 2 times. 100 μl of 0.05M ABC (ammonium bicarbonate) solution was added, mixed with the liquid in the ultrafiltration tube, and incubated in the dark for 20 minutes; 14000g was centrifuged for 10 minutes, and the waste liquid in the collection tube was discarded, and this step was repeated 2 times. Mu.l of a 0.05M ABC solution was added, following protein: enzyme=50-100:1, 1. Mu.g/. Mu.l trypsin stock was added, mixed and incubated at 37℃for 15h. The ultrafiltration tube was transferred to a fresh centrifuge tube and centrifuged at 14000g for 10 minutes. 40 μl of ABC solution was added and the mixture centrifuged at 14000g for 10 minutes. The ultrafiltration tube was discarded and the liquid in the centrifuge tube was lyophilized.
And (3) desalting: the above lyophilized samples were dissolved with 20 μl of 0.4% trifluoroacetic acid (TFA); wetting C with 100% acetonitrile ACN 18 Gun head (Zip Tip C) 18 Millipore) once, 10. Mu.L/time; wetting the gun head twice with 50% ACN, 10 μl/time; the Tip tips were equilibrated twice with 10 μl of 0.1% TFA; sucking 10 mu L of sample, and blowing back and forth for 10 times; wash twice with 0.1% TFA, 10 μl/time; elution was performed with 10 μl of 0.1% formic acid/60% ACN; freeze-drying for about 30 minutes; adding 20 μL of 0.1% formic acid for dissolution, taking 1.5 μL of sample, detecting peptide concentration by Nanodrop, transferring the residual sample into a 250 μL upper sample tube of mass spectrum, placing into a 48-hole upper sample plate of nano-upgrading liquid chromatograph, taking 1 μg total amount of polypeptide according to the measured polypeptide concentration, separating by nano-liter liquid chromatograph (EASY-nLC 1000) and LTQ-Orbitrap Elite high resolutionSpectral identification, analysis and retrieval of mass spectrum data were performed using PEAKS Studio 8.0, with the following parameters:
1. search engine name: PEAKS;
2. parent mass error margin: 10.0ppm;
3. fragment mass error margin: 0.05Da;
4. enzyme: trypsin;
5. fixed modification: carboxymethyl;
6. variable modification: oxidation and degradation;
7. database: a custom database containing four of the characteristic polypeptide sequences selected in example 1.
The search results are shown in Table 3, wherein "Y" indicates "Yes" and "N" indicates "No" in whether or not an item is specific.
TABLE 3 Table 3
The mucin-Ackermans exists in the sample, and the corresponding parent ion mass spectrum and the secondary mass analysis spectrum are shown in figures 1-4, wherein figure 1 is a parent ion mass spectrum (A) and a secondary mass analysis spectrum (B) of polypeptide LLDEGQAGDNVGLLLR [ m/z841.949 (z=2) ]; FIG. 2 is a parent ion mass spectrum (A) and a secondary mass analysis spectrum (B) of polypeptide LMPVEDVFSISGR [ m/z 725.367 (z=2) ]; FIG. 3 is a parent ion mass spectrum (A) and a secondary mass analysis spectrum (B) of polypeptide TAAEYDNYR [ m/z 551.739 (z=2) ]; FIG. 4 shows a parent ion mass spectrum (A) and a secondary mass analysis spectrum (B) of the polypeptide VDFNNVPLK [ m/z466.261 (z=2) ].
Example 3: specificity identification-negative
In the embodiment, equipment is adopted for high-resolution mass spectrum of nano liter liquid chromatograph (EASY-nLC 1000) and LTQ-Orbitrap Elite, and all reagents such as ammonia bicarbonate, trypsin, acetonitrile, formic acid and the like need to be purified by chromatography.
The soil samples were subjected to PCR detection using upstream primer 5'-CAGCACGTGAAGGTGGGGAC-3' and downstream primer 5'-CCTTGCGGTTGGCTTCAGAT-3' (Qin Qianqian, zhangling, wang Guoqing. Real-time fluorescent PCR quantitative detection method for feces Akkermansia muciniphila method study [ J ]. University of Sichuan university, medical edition, 2018 (1): 93-97.) in 2018, etc., and confirmed that samples containing no mucin-Ackermans were confirmed.
Separating bacteria in the soil sample by differential centrifugation, and preparing a polypeptide sample according to the following steps: the bacterial samples were isolated by adding a 100% TCA solution at a ratio of 4:1 (V: V) followed by 1M DTT mother liquor to a final concentration of 100mM. Shaking and mixing uniformly. After 1 hour on ice, 14000g was centrifuged for 5 minutes, and the supernatant was discarded. The precipitate was added with 800. Mu.l of acetone at-20℃and the precipitate was vortex-blown off, allowed to stand at 4℃for 10 minutes, centrifuged at 14000g for 5 minutes, and the supernatant was discarded. The precipitate was added with 300. Mu.l of acetone at-20℃and the precipitate was blown off and sonicated for 15 min to aid dissolution, and allowed to stand at-20℃for 30 min. Centrifuge 6500g for 10 min at 4℃and discard the supernatant. This step was repeated 2 times. The precipitate was dried in a metal bath at 95℃for 5-10 minutes to completely remove the acetone. Fresh 8M UA (urea) solution is prepared, 120 mu l of 8M UA is added to each tube, the precipitate is blown off, the solution is placed in a metal bath at 50 ℃ for 10 minutes, 14000g is centrifuged for 10 minutes and then incubated, the supernatant is taken to obtain a bold protein solution, and the protein concentration is measured by Nano-drop. The corresponding protein solution of 300. Mu.g protein per sample was transferred to a 10K ultrafiltration tube (the tube was placed on a collection tube), 200. Mu.l 8M UA was added, 14000g was centrifuged for 10 minutes, and the waste solution in the collection tube was discarded, and this step was repeated 2 times. 100 μl of 0.05M IAA (iodoacetamide) solution was added and mixed with the liquid in the ultrafiltration tube, followed by incubation in the dark for 20 minutes; 14000g was centrifuged for 10 minutes, and the waste liquid in the collection tube was discarded. 100 μl UA was added and 14000g centrifuged for 10 minutes, the waste liquid in the collection tube was discarded, and the procedure was repeated 2 times. 100 μl of 0.05M ABC (ammonium bicarbonate) solution was added, mixed with the liquid in the ultrafiltration tube, and incubated in the dark for 20 minutes; 14000g was centrifuged for 10 minutes, and the waste liquid in the collection tube was discarded, and this step was repeated 2 times. Mu.l of a 0.05M ABC solution was added, following protein: enzyme=50-100:1, 1. Mu.g/. Mu.l trypsin stock was added, mixed and incubated at 37℃for 15h. The ultrafiltration tube was transferred to a fresh centrifuge tube and centrifuged at 14000g for 10 minutes. 40 μl of ABC solution was added and the mixture centrifuged at 14000g for 10 minutes. The ultrafiltration tube was discarded and the liquid in the centrifuge tube was lyophilized. And (3) desalting: the above-mentioned materials are mixedThe lyophilized samples were dissolved with 20 μl of 0.4% trifluoroacetic acid (TFA); wetting C with 100% acetonitrile ACN 18 Gun head (Zip Tip C) 18 Millipore) once, 10. Mu.L/time; wetting the gun head twice with 50% ACN, 10 μl/time; the Tip tips were equilibrated twice with 10 μl of 0.1% TFA; sucking 10 mu L of sample, and blowing back and forth for 10 times; wash twice with 0.1% TFA, 10 μl/time; elution was performed with 10 μl of 0.1% formic acid/60% ACN; freeze-drying for about 30 minutes; adding 20 mu L of 0.1% formic acid for dissolution, taking 1.5 mu L of sample, detecting peptide concentration by Nanodrop, transferring the residual sample into a 250 mu L upper sample tube of a mass spectrum, placing the sample into a 48-hole upper sample plate of a nano-upgrading liquid chromatograph, separating 1 mu g total amount of polypeptide by a nano-liter liquid chromatograph (EASY-nLC 1000) according to the measured polypeptide concentration, and identifying by LTQ-Orbitrap Elite by high resolution mass spectrum, wherein the mass spectrum data is analyzed and searched by PEAKS Studio 8.0, and the search parameters are as follows:
1. search engine name: PEAKS;
2. parent mass error margin: 10.0ppm;
3. fragment mass error margin: 0.05Da;
4. enzyme: trypsin;
5. fixed modification: carboxymethyl;
6. variable modification: oxidation and degradation;
7. database: a custom database comprising four characteristic polypeptide sequences.
Search results: no corresponding polypeptides were retrieved within the corresponding parent ion mass range.
Sequence listing
<110> university of Zhejiang
<120> a characteristic peptide of mucin-Achroman and use thereof
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> mucin-Ackermans (Akkermansia muciniphila)
<400> 1
Leu Leu Asp Glu Gly Gln Ala Gly Asp Asn Val Gly Leu Leu Leu Arg
1 5 10 15
<210> 2
<211> 7
<212> PRT
<213> mucin-Ackermans (Akkermansia muciniphila)
<400> 2
Glu His Ile Leu Leu Ala Arg
1 5
<210> 3
<211> 13
<212> PRT
<213> mucin-Ackermans (Akkermansia muciniphila)
<400> 3
Leu Met Pro Val Glu Asp Val Phe Ser Ile Ser Gly Arg
1 5 10
<210> 4
<211> 7
<212> PRT
<213> mucin-Ackermans (Akkermansia muciniphila)
<400> 4
Gly Thr Val Ala Thr Gly Arg
1 5
<210> 5
<211> 15
<212> PRT
<213> mucin-Ackermans (Akkermansia muciniphila)
<400> 5
His Tyr Ala His Val Asp Cys Pro Gly His Ala Asp Tyr Val Lys
1 5 10 15
<210> 6
<211> 9
<212> PRT
<213> mucin-Ackermans (Akkermansia muciniphila)
<400> 6
Phe Asn Asn Tyr Arg Pro Gln Phe Tyr
1 5
<210> 7
<211> 8
<212> PRT
<213> mucin-Ackermans (Akkermansia muciniphila)
<400> 7
Val Asp Phe Asn Val Pro Leu Lys
1 5
<210> 8
<211> 9
<212> PRT
<213> mucin-Ackermans (Akkermansia muciniphila)
<400> 8
Ile Val Ala Ala Leu Pro Thr Ile Lys
1 5
<210> 9
<211> 9
<212> PRT
<213> mucin-Ackermans (Akkermansia muciniphila)
<400> 9
Thr Ala Ala Glu Tyr Asp Asn Tyr Arg
1 5
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
cagcacgtga aggtggggac 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
ccttgcggtt ggcttcagat 20
Claims (4)
1. A method for detecting mucin-Ackermans (Akkermansia muciniphila) for non-medical diagnosis purposes, characterized in that a liquid chromatography mass spectrometry method is used to detect a sample, if a characteristic peptide of mucin-Ackermans is present in the sample,
the amino acid sequence of the characteristic peptide is LMPVEDVFSISGR,
the sample to be detected is an environmental sample,
the method comprises the following steps: subjecting sample to be tested to enzyme digestion with protein extraction and trypsin, centrifuging and ultrafiltering the enzymolysis solution with ultrafiltration tube with molecular weight cut-off of 3000, collecting filtrate, lyophilizing, desalting, drying eluate containing polypeptide, dissolving with 0.1% formic acid, quantifying polypeptide content, introducing sample, and performing liquid chromatography C 18 And (3) carrying out spectrogram acquisition and identification by column gradient separation and high-resolution mass spectrometry.
2. The method of claim 1, wherein the sample injection amount is 0.5-1 μg and the sample injection flow rate is 10 nL/min when the liquid chromatography mass spectrometry is combined; liquid chromatography C 18 The conditions for column gradient separation were as follows: phase A is an aqueous solution containing 0.1% formic acid and phase B is an acetonitrile solution containing 0.1% formic acid.
3. The method of claim 1, wherein the spectrogram acquisition time is 150 minutes and the nanoliter liquid phase separation gradient is as follows:
4. the method of claim 1, wherein the parameters of the high resolution mass spectrum acquisition are as follows:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111175902.5A CN114106101B (en) | 2021-10-09 | 2021-10-09 | Characteristic peptide of mucin-Achroman and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111175902.5A CN114106101B (en) | 2021-10-09 | 2021-10-09 | Characteristic peptide of mucin-Achroman and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114106101A CN114106101A (en) | 2022-03-01 |
| CN114106101B true CN114106101B (en) | 2023-10-13 |
Family
ID=80441902
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111175902.5A Active CN114106101B (en) | 2021-10-09 | 2021-10-09 | Characteristic peptide of mucin-Achroman and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114106101B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112666270A (en) * | 2020-09-21 | 2021-04-16 | 天津科技大学 | Novel method for detecting staphylococcus aureus and detection kit thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3185014A1 (en) * | 2015-12-23 | 2017-06-28 | Polyquant GmbH | Bladder cancer biomarker proteins |
-
2021
- 2021-10-09 CN CN202111175902.5A patent/CN114106101B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112666270A (en) * | 2020-09-21 | 2021-04-16 | 天津科技大学 | Novel method for detecting staphylococcus aureus and detection kit thereof |
Non-Patent Citations (6)
| Title |
|---|
| Elongation factor Tu [Desulfonema magnum]，GenBank: QTA85354.1;Schnaars,V.等;《NCBI》;ORIGIN部分 * |
| elongation factor Tu, mitochondrial, partial [Tanacetum cinerariifolium]，GenBank: GEU28356.1;Yamashiro,T.等;《NCBI》;origin部分 * |
| MAG TPA: nucleotide exchange factor GrpE [Ruminococcaceae bacterium]，GenBank: HAH79102.1;Parks,D.H.等;《NCBI》;origin部分 * |
| NCBI Reference Sequence: MBQ4613696.1，MAG: nucleotide exchange factor GrpE [Akkermansia sp];Xie,F.;《NCBI》;20210419;全文 * |
| PDZ domain-containing protein 8-like isoform X1 [Hyposmocoma kahamanoa] ，;unknown;《NCBI》;origin部分 * |
| 金黄色葡萄球菌候选特征肽的靶向分析;赵宏等;《分析化学》;20200707;第48卷(第7期);摘要，第二页2.2.2和2.2.3部分 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114106101A (en) | 2022-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110938130B (en) | Bioactive polypeptide RVFQPLPHENKPLTL, and preparation method and application thereof | |
| Mohri | Amino-acid composition of “Tubulin” constituting microtubules of sperm flagella | |
| Cookson et al. | Biological activities and chemical composition of purified tracheal cytotoxin of Bordetella pertussis | |
| CN107163128B (en) | Preparation and application of kappa-casein-derived bioactive peptide | |
| Brückner et al. | Quantification of D-amino acids in human urine using GC-MS and HPLC | |
| CN110627897B (en) | Active peptide for promoting osteoblast proliferation and application thereof | |
| KR950701648A (en) | ANTIBIOTIC CRYPTDIN PEPTIDES AND METHODS OF THEIR USE | |
| CN113564069A (en) | Bifidobacterium longum and bifidobacterium longum exopolysaccharide as well as extraction method and application thereof | |
| CN114106101B (en) | Characteristic peptide of mucin-Achroman and application thereof | |
| CN112745380B (en) | Bioactive peptide with amino acid structure RAGLQFPVGRVH, and preparation method and application thereof | |
| CN109369783A (en) | A kind of polypeptide RDP1 and its method of purification and application | |
| YASUHARA et al. | The studies on the active peptide on smooth muscle in the skin of Rana rugosa, Thr6-bradykinin and its analogous peptide, ranakinin-R | |
| CN111778193A (en) | Lactobacillus johnsonii, adenosine heptapeptide prepared from lactobacillus johnsonii and preparation method of adenosine heptapeptide | |
| CN114106117B (en) | Characteristic peptide of actinomycetes and application thereof | |
| CN113813291B (en) | Preparation method of animal medicinal material freeze-dried powder | |
| Stein | Isolation of natural proteins | |
| CN112285195B (en) | Characteristic glycoprotein marker of milk exosome and characteristic marker separation method of milk exosome | |
| CN114276953A (en) | Lactobacillus plantarum YE4 capable of inhibiting intestinal cell DPP-4 activity and application thereof in relieving diabetes | |
| CN1800208A (en) | Snake venom polypeptide and its preparation method | |
| Sanders | Isolation and characterization of the nonhistone chromosomal proteins of developing avian erythroid cells | |
| CN112661830A (en) | Bioactive peptide with amino acid structure AIRNDEELNKLLGR, and preparation method and application thereof | |
| CN112480232A (en) | Bioactive peptide VSLADLQNDEVAFR, and preparation method and application thereof | |
| CN112500468A (en) | Bioactive peptide RLAFIAHPKLG, and preparation method and application thereof | |
| CN113072627A (en) | MccJ25 mutant and preparation method and application thereof | |
| CN111763243A (en) | Gorgon fish immune active peptide and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |