CN111189940B

CN111189940B - Method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry

Info

Publication number: CN111189940B
Application number: CN202010042707.4A
Authority: CN
Inventors: 许美娟; 陈影; 姜超; 袭晓昀; 吴婷; 吴坚; 张倩; 储继红; 居文政; 戴国梁
Original assignee: Jiangsu Provincial Hospital of Chinese Medicine
Current assignee: Jiangsu Provincial Hospital of Chinese Medicine
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2022-03-22
Anticipated expiration: 2040-01-15
Also published as: CN111189940A

Abstract

The invention discloses a method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry, which comprises the following steps: (1) screening specific polypeptide of soluble epoxide hydrolase from tissue, wherein the amino acid sequence of the specific polypeptide is shown as SEQ ID NO. 1; detecting by a liquid chromatography-mass spectrometry method by using a specific polypeptide as a standard substance and using a mass spectrum isotope internal standard quantitative method, and establishing a standard curve by using the concentration of the standard substance as a horizontal coordinate and the ratio of a quantitative daughter ion peak area to an isotope internal standard peak area as a vertical coordinate; (2) and (3) determining the concentration of the specific polypeptide in the sample by a liquid chromatography-mass spectrometry method, and then converting and calculating to obtain the concentration of the soluble epoxide hydrolase in the sample. The invention selects the peptide segment with stable enzymolysis and high enzymolysis efficiency from the representative characteristic peptide segments with stable properties as the quantitative peptide segment, has the advantages of accuracy, precision, sensitivity and the like, and can effectively carry out accurate quantitative analysis on sEH in rat tissues.

Description

Method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry

Technical Field

The invention belongs to a method for absolutely quantifying protein, and particularly relates to a method for detecting soluble epoxide hydrolase (sEH) by liquid chromatography-mass spectrometry (LC-MS/MS).

Background

Epoxide Hydrolases (EHs) are a class of enzymes commonly found in mammalian, insect, plant, microbial organisms that stereoselectively add water molecules to epoxides to produce the corresponding 1, 2-diols. Soluble epoxide hydrolase (sEH), a member of the family of epoxide hydrolases, was named in 1973 by Gill et al when studying the metabolism of terpenoid epoxides, analogous to the insect juvenile hormone, because it is located primarily in the soluble components of the cell, such as the cytoplasm and the peroxisome. sEH in mammalian systems has an α/β hydrolase fold structure and is capable of hydrolyzing trans-substituted epoxides and a series of aliphatic epoxides converted from fatty acids. These epoxides are important signaling molecules in the body that modulate a range of physiological functions, such as vascular tone, inflammatory responses, angiogenesis and pain.

CYP450 surface oxidase metabolite epoxyeicosatrienoic acids (EETs) of arachidonic acid are substrates of sEH, and have the effects of resisting inflammation, expanding blood vessels, reducing blood pressure, protecting nerves and the like, and the EETs generate dihydroxyeicosatrienoic acids (DHETs) with lower activity after being metabolized by the sEH. In recent years, a large number of studies show that animals inhibiting the activity of sEH or down-regulating the expression of sEH have advantages in various aspects such as inflammation, diabetes, cardiovascular diseases, neuroprotection and the like, and suggest that sEH plays an important role in diseases such as myocardial hypertrophy, diabetes, hypertension, nephropathy, Parkinson, depression and the like, and is a potential therapeutic target of the diseases. sEH has become a research hotspot at this stage.

Currently, methods for measuring sEH protein expression in tissues mainly include immunohistochemistry and Western blotting. However, both methods are semi-quantitative methods that rely on specific antibodies, are poorly specific, and are not amenable to large sample scale assays. With the application of mass spectrometry technology in proteomics, the absolute quantification of a certain protein by LC-MS/MS becomes possible. The specific polypeptide determined by the method is specific to rat sEH enzyme and has strong specificity. In addition, compared with the traditional Western blotting method, the high-throughput characteristic of the mass spectrum greatly shortens the analysis time and improves the working efficiency.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry aiming at the defects of the prior art.

In order to solve the technical problems, the invention discloses a method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry, which comprises the steps of obtaining an amino acid sequence of the soluble epoxide hydrolase from a Uniprot database, and simulating an enzymolysis process of the soluble epoxide hydrolase by Skyline software to obtain a series of peptide fragments. Taking 0.4g of rat tissue, adding 1mL of water for homogenate, centrifuging at 9000g of the homogenate at 4 ℃ for 20min, and taking supernatant to obtain S9; determining the protein concentration of the S9 sample; subjecting the S9 sample to enzymatic digestion; and performing LC-MS/MS detection on the digested S9 sample, and screening to obtain DFLLGAFQMK specific polypeptide with good phase response. Preparing a high-purity standard substance and an isotope internal standard substance of the specific polypeptide, drawing a standard curve by taking the ratio of the peak area of the quantitive ion of the specific polypeptide to the peak area of the isotope internal standard substance as a vertical coordinate, and calculating to obtain the concentration of the enzyme in the sample.

Specifically, the method comprises the following steps:

(1) screening specific polypeptide of soluble epoxide hydrolase from tissue, wherein the amino acid sequence of the specific polypeptide is shown as SEQ ID NO. 1; detecting by a liquid chromatography-mass spectrometry method by using a specific polypeptide as a standard substance and using a mass spectrum isotope internal standard quantitative method, and establishing a standard curve by using the concentration of the standard substance as a horizontal coordinate and the ratio of a quantitative daughter ion peak area to an isotope internal standard peak area as a vertical coordinate;

(2) and (3) determining the concentration of the specific polypeptide in the sample by a liquid chromatography-mass spectrometry method, and then converting and calculating to obtain the concentration of the soluble epoxide hydrolase in the sample.

Wherein the tissue is any one of heart, liver, spleen, lung, kidney, brain, intestine, stomach, muscle, fat, ovary, testis, placenta and blood vessel of rat.

Wherein, the screening method of the specific polypeptide comprises the steps of adding 1mL of water into 0.4g of tissues for homogenate, centrifuging at 9000g of 4 ℃ for 20min, and taking the supernatant, namely an S9 sample; determining the protein concentration of the S9 sample; subjecting the S9 sample to enzymatic digestion; and (3) carrying out liquid chromatography-mass spectrometry detection on the digested S9 sample, and screening a peptide segment which has good signal and good repeatability and is specific to sEH in a BLAST result to obtain the specific polypeptide.

Wherein the enzymatic digestion comprises transferring 160 μ g S9 sample into a centrifuge tube, adding 10 μ L250 mM Dithiothreitol (DTT) aqueous solution and 40 μ L100 mM Ammonium Bicarbonate (ABC) buffer solution, inactivating at 95 deg.C for 10min, adding 20 μ L500mM 1H-indole-3-carboxylic acid (indoleacetic acid) (IAA) aqueous solution, incubating for 30min in the absence of light, adding 500 μ L methanol and 400 μ L deionized water, centrifuging at 16000g 4 deg.C for 5min, removing the supernatant, adding 60 μ L ABC buffer solution and 20 μ L of 160 μ g/mL^-1After digesting an aqueous solution of ammonium bicarbonate with trypsin (Thermo Scientific, USA) at 37 ℃ for 16h, 20. mu.L of internal standard working solution and 10. mu.L of diluent are added, and the mixture is centrifuged at 12000g at 4 ℃ for 10min to obtain a supernatant, namely a digested S9 sample.

Wherein the internal standard working solution is isotope polypeptide of specific polypeptide shown in SEQ ID NO.1, and the concentration is 200 ng-mL^-1。

The preparation method of the internal standard working solution comprises the following steps:

(i) mu.L of 2.5 mg/mL was diluted with the diluent^-1The internal standard polypeptide stock (namely an isotope internal standard stock with the sequence of DFLLGAFQMK) is diluted to 1mL to obtain 10 mug. multidot.mL^-1The internal standard diluent of (4);

(ii) 20. mu.L of 10. mu.g/mL was diluted with the diluent^-1Diluting the internal standard diluent to 1mL to obtain 200 ng/mL^-1The internal standard working solution of (4).

Among them, the method for determining the protein concentration of the S9 sample is the BCA method.

The preparation method of the standard substance comprises the following steps:

(a) preparing a specific polypeptide standard curve working solution: weighing 0.99mg of specific polypeptide standard substance shown as SEQ ID NO.1, diluting to 1mL with diluent, and mixing to obtain 0.99 mg/mL^-1Specific polypeptide standard curve stock solution; stock solution was diluted with diluent to a concentration of 1. mu.g.mL^-1(ii) a Diluting with diluent in turn to make into 5, 20, 50, 100, 200 and 400 ng/mL respectively^-1The specific polypeptide standard of (1);

(b) preparation of a standard curve: adding 10 μ L250 mM dithiothreitol aqueous solution and 40 μ L100 mM ammonium bicarbonate buffer solution into 160 μ g S9 sample, inactivating at 95 deg.C for 10min, adding 20 μ L500mM 1H-indole-3-carboxylic acid (indoleacetic acid) aqueous solution, incubating for 30min in the absence of light, adding 500 μ L methanol and 400 μ L deionized water, centrifuging at 16000g 4 deg.C for 5min, removing the supernatant, adding 60 μ L ammonium bicarbonate buffer solution and 20 μ L ammonium bicarbonate buffer solution with concentration of 160 μ g.mL^-1Digesting the trypsin ammonium bicarbonate aqueous solution at 37 ℃ for 16h, adding 20 mu L of internal standard working solution and 10 mu L of standard curve working solution with each concentration obtained in the step (a), centrifuging at 12000g and 4 ℃ for 10min, and taking supernatant to obtain the compound.

Wherein the diluent is an aqueous solution containing formic acid and acetonitrile; wherein the volume fraction of formic acid is 0.5%, and the volume fraction of acetonitrile is 80%.

Wherein, the specific chromatographic conditions for the liquid chromatography-mass spectrometry are as follows:

(1) conditions of liquid chromatography

A chromatographic column: agilent SB-C18, 4.6X 100mm, 1.8 μm; protection of the column: agilent ZORAX SB-C18, 2.1 × 12.5mm, 5 μm; mobile phase: a: b-water (0.1% FA in water (v/v)): acetonitrile, gradient elution; column temperature: 40 ℃;

(2) conditions of Mass Spectrometry

The compound detection was performed using an API4000 MS/MS mass spectrometer from AB. Detection is carried out by adopting an ESI ion source and a positive ion MRM mode, and the ion source parameters are as follows: CAD: 12; and (4) CUR: 20; GS 1: 40; GS 2: 60, adding a solvent to the mixture; IS: 5500; TEM: 500, a step of; EP: 10; CXP: 9;

the same parent ion and different daughter ions can help to judge the peak position of the sample peak. Thus, the table lists 2 ion pairs for qualitative (to aid in determining peak position) and 1 ion pair for quantitative (for calculation).

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the invention establishes a method for measuring the concentration of soluble epoxide hydrolase sEH in rat tissues, and has the advantages of accuracy, precision and high sensitivity.

(2) By the method, the protein concentration of sEH in heart, liver, kidney and other tissues of a rat can be measured accurately in a large scale, and support is provided for researching the occurrence and development of diseases and the action mechanism of medicaments.

Drawings

FIG. 1 is an LC-MS/MS chromatogram of sEH in tissue, where A is blank; b is the sample with the lowest limit of quantitation of the standard curve (0.5 ng. mL)^-1Concentration); c is actual kidney tissue sample; wherein 1 represents sEH-specific polypeptide DFLLGAFQMK; 2 represents an isotopic internal standard DFLLGAFQMK of an sEH-specific polypeptide; 3 represents a peptidomimetic segment EKDFLLGAFQMK of a sEH-specific polypeptide.

FIG. 2 is an internal standard calibration curve of the quantification method of the embodiment, wherein the characters in the graph show that the correlation coefficient r of the standard curve is greater than 0.999, which indicates that the correlation coefficient r is in the range of 0.5-40.0 ng.mL^-1The linearity within the range is good.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: determination of sEH protein concentration in rat Heart, liver and Kidney tissues

First, experimental material and instrument

Materials: DFLLGAFQMK standard peptide fragment and EKDFLLGAFQMK peptidomimetic: jiangsu Jinsrie Biotech limited, the content is more than 98.5%; DFLLGAFQMK stock solution of isotopic peptide fragments: thermo Scientific, usa, content greater than 95%; BCA kit, DDT, IAA, and pancreatin: thermo Scientific; acetonitrile and methanol: merck Company, HPLC grade; formic acid: ACS corporation, HPLC grade; the water was prepared from a Millipore Milli-Q Advantage A10 ultra pure water machine.

The instrument comprises the following steps: API4000 LC-MS/MS LC-MS comprising: (1) pump Agilent 1260G 1312A; (2) column Oven Agilent 1260G 1316A; (3) auto Sampler Agilent 1260G 1367E; (4) mass spectrometer API 4000; (5) value Valco 2-Position, controlled by Analyst 1.6 Software; WH-2 micro vortex mixer (Shanghai province of analytical instruments); MS-100 constant temperature mixer (Hangzhou Osheng instruments Co., Ltd.); WD-2102B full-automatic enzyme marker (Beijing, six Biotech Co., Ltd.); CPA225D electronic balance (sidures, germany); millipore Drict-Q5 Water purifier (Millipore, France); biofuge PrimoR refrigerated high speed centrifuge (Heraeus, Germany).

Second, liquid condition

1. Conditions of liquid chromatography

A chromatographic column: agilent SB-C18, 4.6X 100mm, 1.8 μm; protection of the column: agilent ZORAX SB-C18, 2.1 × 12.5mm, 5 μm; mobile phase: a: b-water (0.1% FA in water (v/v)): acetonitrile, gradient elution, see table 1; column temperature: 40 ℃; the amount of sample was 7. mu.L.

TABLE 1 mobile phase gradient elution parameters

2. Conditions of Mass Spectrometry

The compound detection was performed using an API4000 MS/MS mass spectrometer from AB. Detection is carried out by adopting an ESI ion source and a positive ion MRM mode, and the ion source parameters are as follows: CAD: 12; and (4) CUR: 20; GS 1: 40; GS 2: 60, adding a solvent to the mixture; IS: 5500; TEM: 500, a step of; EP: 10; CXP: 9.

TABLE 2 Mass Spectrometry parameters

Thirdly, an experimental process:

1. preparing a polypeptide standard curve working solution:

precisely weighing 0.99mg of polypeptide standard substance shown as SEQ ID NO.1 into a 1mL volumetric flask, adding diluent (80 v/v% acetonitrile solution containing 0.5 v/v% FA) to a constant volume to scale, and mixing to obtain 0.99 mg/mL^-1Polypeptide standard curve stock. Precisely measuring appropriate amount of stock solution, and diluting with diluent to concentration of 1 μ g/mL^-1A polypeptide standard product working solution; diluting with diluent in turn to make into 5, 20, 50, 100, 200 and 400 ng/mL respectively^-1The polypeptide standard curve working solution is stored in a refrigerator at the temperature of minus 20 ℃ for later use.

2. Preparing a polypeptide quality control working solution:

precisely weighing 1.02mg of polypeptide standard product into a 1mL volumetric flask, adding diluent to a constant volume to scale, and uniformly mixing to obtain 1.02 mg/mL^-1Polypeptide quality control stock solution. Precisely measuring appropriate amount of quality control stock solution, and diluting with diluent to concentration of 300, 60 and 6 ng/mL^-1Respectively obtaining polypeptide quality control working solution with high, medium and low concentration, and storing in a refrigerator at-20 ℃ for later use.

3. Preparing an internal standard working solution:

precisely measuring internal standard polypeptide stock solution (2.5 mg. mL)^-1) Diluting 4 μ L with diluent to 1mL, and mixing to obtain 10 μ g/mL^-1Precisely measuring 20 mu L of the internal standard diluent, adding the diluent to dilute to 1mL, and uniformly mixing to obtain 200ng & mL^-1The internal standard working solution is stored in a refrigerator at the temperature of minus 20 ℃ for standby.

4. Collecting a tissue sample:

6 Wistar rats (female half) are killed by decapitation, the tissues of the heart, the liver and the kidney are immediately taken out, washed by normal saline, subjected to water absorption, quick-frozen by liquid nitrogen, and then transferred to-80 ℃ for storage for later use.

5. Preparation of tissue S9 sample:

weighing about 0.4g of each tissue, cutting the tissue into pieces by using an ophthalmic scissors, adding 1mL of ultrapure water to prepare tissue homogenate, centrifuging the tissue homogenate for 20min at the temperature of 4 ℃ and 9000g of the tissue homogenate, and taking supernatant to obtain S9 sample solution of each tissue.

BCA assay determination of protein concentration of S9 sample solution:

an appropriate amount of the S9 sample was diluted 80-fold with ultrapure water, and then the protein concentration of the S9 sample was measured according to the BCA kit.

Enzymatic digestion of S9 samples

Mu.g of tissue S9 sample was transferred to a centrifuge tube, 10. mu.L of LDTT (250mM) and 40. mu.L of LABC buffer (100mM) were added, inactivation was performed at 95 ℃ for 10min, 20. mu.L of IAA (500mM) was added, after incubation for 30min in the dark, 500. mu.L of methanol and 400. mu.L of deionized water were added, followed by centrifugation at 16000g for 5min at 4 ℃. After removing the supernatant, 60. mu.L of LABC buffer and 20. mu.L of 160. mu.g/mL of buffer were added^-1Digesting the trypsin ammonium bicarbonate aqueous solution for 16h at 37 ℃, adding 20 mu L of internal standard working solution and 10 mu L of diluent, and centrifuging at 12000g and 4 ℃ for 10min, wherein the volume of the digested solution is the supernatant, and the total volume is 110 mu L; mu.L of the supernatant was measured by LC-MS/MS method.

8. Preparation of tissue standard curve and quality control sample

Transferring 160 mu g of mouse heart tissue S9 sample into a centrifuge tube, carrying out enzymolysis for 16h at 37 ℃ under the item of enzymatic digestion of S9 sample, adding 20 mu L of internal standard working solution and 10 mu L of standard curve working solution or quality control working solution with various concentrations, centrifuging for 10min at 12000g and 4 ℃, taking 7 mu L of supernatant, and determining by an LC-MS/MS method.

9. Method verification

(1) The specificity is as follows:

mu.g of mouse heart tissue S9 sample is transferred into a centrifuge tube, digested for 16h at 37 ℃ by enzymolysis under the item of 'enzymatic digestion of S9 sample', 20 mu.L of internal standard working solution and 10 mu.L of diluent are added, centrifugation is carried out for 10min at 12000g and 4 ℃, and the supernatant is taken for determination by an LC-MS/MS method.

The results show that under the selected LC-MS/MS conditions (as shown in FIG. 1), the retention time of the polypeptide of the sEH sample and the retention time of the internal standard are both about 12.8min, the S9 sample of the mouse heart has no impurity peak to influence the accuracy of the determination, and the determination method can be used for determining the concentration of the sEH in rat tissues.

(2) Linear range

Mu.g of mouse heart tissue S9 sample is transferred into a centrifuge tube, and after digestion is carried out for 16h at 37 ℃ by enzymolysis under the item of 'enzymatic digestion of S9 sample', 20 mu.L of internal standard working solution and 10 mu.L of standard curve working solution with each concentration are added, centrifugation is carried out for 10min at 12000g and 4 ℃, and 7 mu.L of supernatant is taken and determined by an LC-MS/MS method. Sample polypeptide and internal standard peak areas were recorded. Taking the polypeptide concentration C of the sample as an X axis, and taking the peak area ratio f of the peak area of the sample to the peak area of the internal standard as a Y axis to carry out weight regression (the weight coefficient is 1/C)²) And calculating the r value. The results show (FIG. 2) that the concentration of the polypeptide in the tissue is 0.5-40.0 ng/mL^-1Within the range, the ratio of the concentration to the peak area has a good linear relation, and the r value of the polypeptide is more than 0.999.

(3) Accuracy and precision

The preparation of peptide-containing concentrations of 0.6, 6 and 30 ng/mL were performed according to the "preparation of tissue Standard Curve and quality control sample^-15 samples were prepared for each concentration and 3 batches were tested, each following a standard curve. And recording the chromatogram and the peak areas of the sample polypeptide and the internal standard. And (4) calculating the peak area ratio f of the sample to the internal standard, and substituting the value f into the following standard curve to obtain the actually measured concentration and the accuracy thereof. The results are shown in Table 3, the accuracy of the actually measured concentration of the quality control samples with low, medium and high polypeptide concentrations is 95.50-108.3 percent in batches, the RSD is 4.68-7.34 percent, the accuracy of the batches is 100.1-105.3 percent and the RSD is 5.19-5.81 percent; i.e., the method has within acceptable limits both batch-to-batch precision and accuracy.

TABLE 3 results of accuracy and precision

(4) Matrix effect

Transferring 160 mu g of mouse heart tissue, rat liver tissue and rat kidney tissue S9 samples into a centrifuge tube, carrying out enzymolysis for 16h at 37 ℃ under the condition of enzymatic digestion of an S9 sample, adding 20 mu L of low-concentration and high-concentration internal standard working solution and 10 mu L of diluent, centrifuging for 10min at 12000g and 4 ℃, taking 7 mu L of supernatant, and determining by an LC-MS/MS method, or adding 20 mu L of low-concentration and high-concentration internal standard working solution and 10 mu L of diluent into 60 mu L of ABC, taking 7 mu L of supernatant after mixing uniformly, and determining by an LC-MS/MS method. The results are shown in table 4, where the matrix effects of low and high concentrations of the internal standard polypeptide isotope approach those in the above tissues.

TABLE 4 results of matrix Effect (%)

(5) Efficiency of enzymolysis

160 mu g of rat heart, liver and kidney tissue S9 sample is transferred into a centrifuge tube, after digestion is carried out for 12, 14, 16 and 18h at 37 ℃ by enzymolysis under the item of 'enzymatic digestion of S9 sample', 20 mu L of internal standard working solution and 10 mu L of diluent are added, centrifugation is carried out for 10min at 12000g and 4 ℃, 7 mu L of supernatant is taken and determined by LC-MS/MS method, the generated amount of 18h is taken as reference, the result is shown in Table 5, and the generated amount of polypeptide is more than 99% when each tissue is digested for 16 h.

160 mu g of rat kidney tissue S9 sample is transferred into a centrifuge tube, 5ng of peptide mimetic sample is added at the same time, after digestion is carried out for 16h at 37 ℃ under the condition of enzymatic digestion of the S9 sample, 20 mu L of internal standard working solution and 10 mu L of diluent are added, centrifugation is carried out for 10min at 12000g and 4 ℃, 7 mu L of supernatant is taken and determined by an LC-MS/MS method, and the result shows that the sample peak of peptide mimetic can not be detected in the sample, so that the enzymolysis efficiency of the sample is inferred to be 100%.

TABLE 5 enzymatic time results%

11. Measurement results

The concentrations of sEH in heart, liver and kidney tissue samples of Wistar rats are shown in table 6.

Wherein the molecular weight of the specific polypeptide is 1169.6 g.mol^-1The volume of the solution after digestion was 110. mu.L, the amount of S9 used was 160. mu.g, the weight of the heart, liver and kidney tissues obtained was 0.4g, and the total amount of S9 protein was the product of the concentration of S9 measured by BCA method and the volume of S9 prepared.

TABLE 6 expression level of sEH in Wistar rat tissue (n ═ 6)

Fourth, discuss

In this study, the LC-MS/MS method was used to detect soluble epoxide hydrolase sEH in rat tissues. At present, a method for detecting soluble epoxide hydrolase sEH in tissues is mainly a Western blot method, but the method can only semi-quantitatively detect the absolute concentration of the protein in the tissues, and is not suitable for detecting the concentration of a large batch of samples. The invention adopts a target quantitative proteomics method, not only can detect the absolute concentration of sEH in rat tissues, but also can be used for detecting mass samples.

The present invention provides a method and a method for detecting soluble epoxide hydrolase sEH by liquid chromatography-mass spectrometry, and a plurality of methods and ways for implementing the method, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should be considered as within the protection scope of the present invention.

Sequence listing

<110> Jiangsu province Chinese medicine institute

<120> method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry

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<170> SIPOSequenceListing 1.0

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<213> Standard Peptide (Standard Peptide)

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Asp Phe Leu Leu Gly Ala Phe Gln Met Lys

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<213> Peptidomimetics

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Glu Lys Asp Phe Leu Leu Gly Ala Phe Gln Met Lys

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Claims

1. A method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry is characterized by comprising the following steps:

(2) measuring the concentration of the specific polypeptide in the sample by a liquid chromatography-mass spectrometry method, and then converting and calculating to obtain the concentration of the soluble epoxide hydrolase in the sample;

(1) conditions of liquid chromatography

A chromatographic column: agilent SB-C18, 4.6X 100mm, 1.8 μm; protection of the column: agilent ZORAX SB-C18, 2.1 × 12.5mm, 5 μm; mobile phase: a: b = 0.1% v/vAqueous formic acid solution: acetonitrile, gradient elution; column temperature: 40 ℃;

(2) conditions of Mass Spectrometry

The compound detection is carried out by adopting an API4000 MS/MS mass spectrometer of AB company, ESI ion source and positive ion MRM mode detection are adopted, and the ion source parameters are as follows: CAD: 12; and (4) CUR: 20; GS 1: 40; GS 2: 60, adding a solvent to the mixture; IS: 5500; TEM: 500, a step of; EP: 10; CXP: 9;

。

2. the method of claim 1, wherein the tissue is any one of heart, liver, spleen, lung, kidney, brain, intestine, stomach, muscle, fat, ovary, testis, placenta and blood vessel of rat.

3. The method of claim 1, wherein the specific polypeptide is selected by adding 1mL of water homogenate to 0.4g of tissue, centrifuging at 9000g of the homogenate at 4 ℃ for 20min, and collecting the supernatant as an S9 sample; determining the protein concentration of the S9 sample; subjecting the S9 sample to enzymatic digestion; and (3) carrying out liquid chromatography-mass spectrometry detection on the digested S9 sample, and screening to obtain the specific polypeptide.

4. The method of claim 3, wherein the enzymatic digestion comprises adding 10 μ L of 250mM aqueous dithiothreitol solution and 40 μ L of 100mM ammonium bicarbonate buffer to 160 μ g S9 samples, inactivating at 95 ℃ for 10min, adding 20 μ L of 500mM aqueous 1H-indole-3-carboxylic acid (indoleacetic acid) solution, incubating for 30min in the absence of light, adding 500 μ L of methanol anddeionized water 400 μ L, centrifuging at 16000g and 4 deg.C for 5min, removing supernatant, adding ammonium bicarbonate buffer 60 μ L and ammonium bicarbonate buffer 20 μ L with concentration of 160 μ g/mL^-1After the trypsin ammonium bicarbonate aqueous solution is digested for 16h at 37 ℃, 20 mu L of internal standard working solution and 10 mu L of diluent are added, centrifugation is carried out for 10min at 12000g and 4 ℃, and supernatant is taken, namely the digested S9 sample.

5. The method of claim 4, wherein the internal standard working solution is an isotope polypeptide of the specific polypeptide shown in SEQ ID NO.1, and the concentration of the isotope polypeptide is 200 ng-mL^-1。

6. The method according to claim 5, wherein the preparation method of the internal standard working solution comprises the following steps:

(i) mu.L of 2.5 mg/mL was diluted with the diluent^-1Diluting the stock solution of the internal standard polypeptide to 1mL to obtain 10 mu g/mL^-1The internal standard diluent of (4);

7. The method according to claim 4, wherein the method for determining the protein concentration of the S9 sample is a BCA method.

8. The method of claim 1, wherein the standard is prepared by a method comprising the steps of:

(b) preparation of a standard curve:adding 10 μ L250 mM dithiothreitol aqueous solution and 40 μ L100 mM ammonium bicarbonate buffer solution into 160 μ g S9 sample, inactivating at 95 deg.C for 10min, adding 20 μ L500mM 1H-indole-3-carboxylic acid (indoleacetic acid) aqueous solution, incubating for 30min in dark, adding 500 μ L methanol and 400 μ L deionized water, centrifuging at 16000g 4 deg.C for 5min, removing the upper layer liquid, adding 60 μ L ammonium bicarbonate buffer solution and 20 μ L ammonium bicarbonate buffer solution with concentration of 160 μ g.mL^-1Digesting the trypsin ammonium bicarbonate aqueous solution at 37 ℃ for 16h, adding 20 mu L of internal standard working solution and 10 mu L of standard curve working solution with each concentration obtained in the step (a), centrifuging at 12000g and 4 ℃ for 10min, and taking supernatant to obtain the compound.

9. The method according to claim 4, 6 or 8, wherein the diluent is an aqueous solution containing formic acid and acetonitrile; wherein the volume fraction of formic acid is 0.5%, and the volume fraction of acetonitrile is 80%.