CN109187403B

CN109187403B - Method for detecting differential protein of effect of 5-phenyl-1- (p-methylphenyl) -1H-triazole and serum

Info

Publication number: CN109187403B
Application number: CN201811226159.XA
Authority: CN
Inventors: 何文英; 吴禄勇; 陈光英; 冯华杰; 刘荣强; 段红叶
Original assignee: Hainan Normal University
Current assignee: Hainan Normal University
Priority date: 2018-10-19
Filing date: 2018-10-19
Publication date: 2021-07-23
Anticipated expiration: 2038-10-19
Also published as: CN109187403A

Abstract

The invention relates to a method for detecting differential protein of 5-phenyl-1- (p-methylphenyl) -1H-triazole and serum action, which comprises the following steps: mixing a serum sample with a 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound; removing the abundant protein; bradford quantifies protein concentration; performing a two-dimensional electrophoresis experiment; processing film image and identifying mass spectrum. The invention also provides a preparation method of the 5-phenyl-1- (p-methylphenyl) -1H-triazole.

Description

Method for detecting differential protein of effect of 5-phenyl-1- (p-methylphenyl) -1H-triazole and serum

Technical Field

The invention belongs to the field of organic synthesis and biochemistry, and particularly relates to a method for detecting differential protein of 5-phenyl-1- (p-methylphenyl) -1H-triazole and serum effects.

Background

The 1,2, 3-triazole compound has a unique five-membered aromatic nitrogen-containing heterocyclic structure, can be conjugated or combined with other substituents to form various derivatives with pharmaceutical activity, has low toxicity and good biological activity, has wide application in many fields of material chemistry, pharmaceutical chemistry, organic chemistry, organometallic chemistry and the like, and is favored by researchers. Proteomics is a subject for understanding life mechanism at protein level, and its academic ideas and related technical methods have been widely applied in various fields of life science, involve many important biological phenomena, and have become one of effective methods for diagnosing, treating and searching drug targets of human serious diseases. The target of action of small molecule drugs is usually protein, and proteomics is a means for detecting the whole target proteome by using chemical small molecules, and the change of the proteome is observed from the whole level, so that the differential expression protein is discovered, the biological function of the protein and the action mechanism of the protein and the small molecules are researched, and a new drug target is found or the small molecules with medicinal value are discovered.

The protein in human blood is almost related to all cells, tissues and organs in the body, and the dynamic change of the blood protein can directly reflect the health condition of the body, so that the protein is the most valuable specimen for researching various disease related markers. The compound 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole is cultured in vitro by combining serum and the compound under the condition of simulating human physiological temperature, and the research on the serum proteome in the presence of the compound is carried out by combining a proteomic research method with MALDI-TOF-MS technology, so that differential expression protein is analyzed, specific serum protein markers of specific diseases are searched, or new disease-related proteins are found, a new basis is provided for the diagnosis or treatment of the diseases, and a more visual theoretical basis is provided for the development of a new medicament possibly serving as the 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole.

Disclosure of Invention

The invention provides a method for detecting differential protein by the action of 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole and serum, which is characterized by comprising the following steps:

(1) uniformly mixing the serum protein with a 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound solution to obtain a sample a, and culturing at constant temperature to obtain a sample b;

(2) removing the abundant protein in the sample b to obtain a sample c

(3) The protein concentration of sample c was quantified by the Bradford method.

(4) Performing a two-dimensional electrophoresis experiment:

firstly, carrying out first-direction isoelectric focusing according to standard hydration loading with the loading amount of 1.3mg of protein of each strip and the total volume of loaded samples of 455 mu L;

after the first direction isoelectric focusing is finished, balancing the adhesive tape and transferring the adhesive tape to polyacrylamide gel to perform a second vertical electrophoresis experiment;

thirdly, dyeing, decoloring, storing and scanning by using Coomassie brilliant blue after electrophoresis is finished;

(5) film image processing and mass spectrometric identification

Gel image analysis, screening out protein points with abnormal expression and marking;

secondly, carrying out enzymolysis and mass spectrum identification on the differential protein points;

(6) results

20 differentially expressed protein spots were obtained using ImageMaster5.0 gel image analysis software, and information on differentially expressed proteins Nos. 1-16 and 18-20 was determined from the Blast search by NCBInr (see Table below).

The 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound solution in the step (1) is a methanol solution of the 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound, and the concentration of the 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound in the sample a is 2.0X 10^-3mol/L; constant-temperature culture: the temperature is 37 ℃, the rotating speed r is 280rpm, and the time is 1 h; the serum protein is preferably human serum protein;

the method for removing the high-abundance protein in the step (2) preferably removes the high-abundance protein according to the requirement of an AndyBio kit;

the specification of the rubber strip in the step (4) is 24cm, and the pH value of the IPG of the nonlinear IPG rubber strip is 3-10 NL.

Another embodiment of the present invention provides the use of 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole for determining differential proteins in their interaction with serum proteins.

Another embodiment of the present invention provides a method for preparing 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole, characterized by comprising the steps of:

dissolving 1-azido-4-methylbenzene and styrene in an organic solvent at room temperature, and adding SmI₂Stirring for 2 hours to obtain 5-phenyl-1- (p-methyl)Phenyl) -1H-1,2, 3-triazole;

the organic solvent is a mixed solution of THF and HMPA in a volume ratio of 1: 1; 1-azido-4-methylbenzene, styrene, SmI₂In a molar ratio of 1: 1.0-1.2: 2.0.

compared with the prior art, the invention has the advantages that: (1) the invention establishes the two-dimensional electrophoresis research method for detecting the differential protein of the 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound and human serum, and has the advantages of short sample processing time, simple operation, clear obtained spectrum and good repeatability; 20 differential expression proteins are obtained in total, 19 differential expression proteins are successfully identified, and a scientific and reasonable theoretical basis is laid for further understanding the related blood protein information and related metabolic networks in the process that 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole as an active compound micromolecule participates in the transportation and metabolism process, more deeply researching and developing and utilizing 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole; (2) the invention provides a method for preparing SmI₂The novel method for preparing 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole by catalyzing 1-azido-4-methylbenzene and styrene is simple and convenient to operate and high in yield.

Drawings

FIG. 1 is a structural diagram of 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole;

FIG. 2 is a two-dimensional electropherogram of a human serum sample;

FIG. 3 is a two-dimensional electropherogram of interaction of human serum sample with 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound;

FIG. 4 is a primary mass spectrum of protein No. 1;

FIG. 5 is a secondary map of molecular weight 1994.1335(+1) of the peptide fragment of protein No. 1;

FIG. 6 is a first order mass spectrum of protein No. 2;

FIG. 7 is a secondary map of molecular weight 1478.7638(+1) of the No. 2 protein peptide fragment;

FIG. 8 is a first order mass spectrum of protein No. 3;

FIG. 9 is a secondary map of molecular weight 1546.6819(+1) of the peptide fragment of protein No. 3;

FIG. 10 is a primary mass spectrum of protein No. 4;

FIG. 11 is a secondary map of the molecular weight of the peptide fragment of protein No. 4 at 2680.5447(+ 1);

FIG. 12 is a primary mass spectrum of protein No. 5;

FIG. 13 is a secondary map of molecular weight 1773.8827(+1) of the peptide fragment of protein No. 5;

FIG. 14 is a first order mass spectrum of protein No. 6;

FIG. 15 is a secondary map of the molecular weight of the peptide fragment of protein No. 6 at 2186.1663(+ 1);

FIG. 16 is a primary mass spectrum of protein No. 7;

FIG. 17 is a secondary map of the molecular weight of the peptide fragment of protein No. 7 at 1646.9363(+ 1);

FIG. 18 is a first order mass spectrum of protein No. 9;

FIG. 19 is a secondary map of the molecular weight of the No. 9 protein peptide fragment 2296.2661(+ 1);

FIG. 20 is a first order mass spectrum of protein No. 10;

FIG. 21 is a secondary map of the molecular weight of the No. 10 protein peptide fragment of 2296.2036(+ 1);

FIG. 22 is a first order mass spectrum of protein No. 11;

FIG. 23 is a secondary map of the molecular weight of the peptide fragment of protein No. 11 at 1874.9916(+ 1);

FIG. 24 is a primary mass spectrum of protein No. 12;

FIG. 25 is a secondary map of molecular weight 1389.8960 of protein peptide fragment of protein No. 12;

FIG. 26 is a first order mass spectrum of protein No. 13;

FIG. 27 is a secondary map of molecular weight 1902.0244 of No. 13 protein peptide fragment;

FIG. 28 is a first order mass spectrum of protein No. 14;

FIG. 29 is a secondary map of molecular weight 980.54681 of No. 14 protein peptide fragment;

FIG. 30 is a first order mass spectrum of protein No. 15;

FIG. 31 is a secondary map of molecular weight 1215.8031 of peptide fragment of protein No. 15;

FIG. 32 is a first order mass spectrum of protein No. 16;

FIG. 33 is a secondary map of molecular weight 2730.6677 of No. 16 protein peptide fragment;

FIG. 34 is a first order mass spectrum of protein No. 18;

FIG. 35 is a secondary map of molecular weight 1406.7456 of protein peptide fragment of No. 18 protein;

FIG. 36 is a first order mass spectrum of protein No. 19;

FIG. 37 is a secondary map of molecular weight 1311.8309 of peptide fragment of protein No. 19;

FIG. 38 is a first mass spectrum of protein No. 20;

FIG. 39 is a secondary map of molecular weight 1716.9952 of No. 20 protein peptide fragment.

Detailed Description

In order to facilitate a further understanding of the invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the present invention and are not intended to limit the scope or the principle of the present invention, and the embodiments of the present invention are not limited to the following.

EXAMPLE I blood sample Material

Healthy human blood samples are selected for 100 cases of men and women, and the average age is 17 plus or minus 1 year. All blood samples are collected in the early morning under an empty stomach state, venous blood of a physical examiner is extracted and placed in a clean and sterile test tube, the test tube is quickly centrifuged and layered after standing for 2 hours, and the test tube is stored in an ultra-low temperature refrigerator at minus 80 ℃.

Example II serum samples and 1,2, 3-triazole Compound were mixed and cultured

Randomly sampling 12 blood samples (6 male and female cases), dissolving and collecting upper layer serum protein, adding 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound stock solution (5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole in methanol) to make its concentration in serum sample 2.0 × 10^-3And (4) mixing the components in mol/L and h uniformly. 2ml of the mixed serum sample and 2ml of the control serum sample were placed in a 37 ℃ incubator and incubated at 280rpm for 1 hour.

Example III removal of high abundance proteins in serum

The incubated serum samples were all handled as required by the AndyBio kit. The method comprises the following specific steps:

1) 100ul of reagent A in ice bath was added to 20ul of serum sample and mixed well.

2) Refrigerating in a refrigerator at-20 deg.C for 90 min.

3) The sample was removed and centrifuged at 15000g at 4 ℃ for 20 min.

4) The supernatant was carefully removed.

5) 1ml of reagent B in ice bath was taken out to the precipitate and mixed well.

6) The mixture was placed in ice water for 15 min.

7) The mixture was then centrifuged at 15000g at 4 ℃ for 20 min.

8) The supernatant was carefully removed.

9) The precipitated sample was air dried naturally.

10) And adding a protein lysate according to the proportion of 10mg/mL to completely dissolve the air-dried sample, wherein the obtained white solution is the sample from which the high-abundance protein is removed.

Example four measurement of protein concentration in sample solution by Bradford method

1) A Bradford solution was prepared.

2) Preparing a standard protein solution, accurately weighing a bovine serum albumin sample, and preparing the bovine serum albumin sample into a 1mg/mL solution by using ultrapure water.

3) The standard protein stock was diluted with Bradford solution to a range of concentrations of 0, 2, 4, 6, 8, 10. mu.g/mL.

4) And measuring the absorbance value of the series of standard protein solutions at 595nm by using an ultraviolet spectrophotometry, and making a standard curve equation according to the standard protein concentration and the corresponding absorbance value.

5) And (3) measuring the absorbance value of the target protein sample solution at 595nm, making a standard curve, and calculating the protein concentration in the sample.

EXAMPLE V two-dimensional electrophoresis Experimental procedure

1) Hydrated loading

And (3) carrying out in-gel hydration sample loading in a disposable hydration disc for 18h at room temperature and 20 ℃ according to the standard that the loading amount of protein of each gel strip is 1.3mg and the total loading volume is 455 mu L, wherein the specification of the gel strip is 24cm, and the pH value of the IPG of the nonlinear IPG gel strip is 3-10 NL.

2) Isoelectric focusing in first dimension

The hydrated gel strip was quickly transferred to an Ettan IPGphor3 focuser at 20 ℃. Run according to the parameters in table 1:

TABLE 1 isoelectric focusing parameters

3) Glue making

Preparing 12.5% of vertical plate polyacrylamide gel, injecting the glue mother liquor into a clean anhydrous glass interlayer, reserving a space of about 1cm at the upper part, and sealing with saturated n-butyl alcohol.

4) Adhesive tape balancing

After the completion of the electropolymerization, the IPG gel strip was shaken for 15min in an equilibrium solution I (0.125 g of dimercaptothreitol was added to each 10ml of the equilibrium buffer solution) and an equilibrium solution II (0.125 g of iodoacetamide was added to each 10ml of the equilibrium buffer solution). The basic formulas of the equilibrium solutions I and II are as follows: PH 8.8, 1.5M tris-HCl, urea 6M, glycerol 30%, SDS 2%, bromophenol blue 0.002%.

5) Second dimension-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

The balanced adhesive tape is transferred to the upper part of polyacrylamide gel, residual air bubbles under the adhesive tape are removed, and the adhesive tape is stabilized by agarose gel sealing liquid. After the agarose is solidified, the gel glass plate is fixed on a vertical electrophoresis mould and is inserted into an electrophoresis tank. The circulating water bath was set at 16 ℃ and run at an electric power of 5W for 1h and an electric power of 7W until the bromophenol blue indicator reached about 5mm from the bottom of the gel.

6) Dyeing and decolorizing

And marking the cut angle of the gel sheet, putting the gel sheet into Coomassie brilliant blue staining solution, and horizontally shaking for about 10 hours. And putting the dyed gel sheet into a decoloring solution, horizontally shaking for 20min, and repeating twice. Then using ultrapure water to elute until the background color of the gel sheet disappears.

Example six image processing and Mass Spectrometry identification

1) Gel image analysis

Scanning a gel map by using an Image Scanner III Scanner, analyzing the scanned Image by using Image Master5.0 analysis software, comparing and screening protein spots with abnormal expression, and marking the protein spots.

2) Making dried colloidal particles

The protein spots matched with the marker proteins were dug out, placed in appropriate EP tubes and labeled. And sequentially adding ultrapure water, a decoloring solution and acetonitrile for washing and decoloring, and placing the obtained colloidal particles on a super clean bench for blowing and drying by small wind.

3) Enzymatic hydrolysis of protein samples

According to the volume of the dry colloidal particles, 5-6 μ L of trypsin solution (20 ng/. mu.L) is added until the colloidal particles are completely covered, and the mixture is placed in a refrigerator at 4 ℃ for 1h to ensure that the colloidal particles fully absorb the enzyme solution. Removing redundant enzyme solution, adding 5-7 mul of trypsin buffer according to the size of colloidal particles, and carrying out enzymolysis for about 13h at 37 ℃. And centrifuging the protein sample subjected to enzymolysis for 5min at normal temperature at 10000g, and collecting the enzymolysis liquid for mass spectrum identification.

4) Mass spectrum identification and database retrieval of target protein

And (3) identifying the differential protein by adopting matrix-assisted laser analysis time-of-flight mass spectrometry, and correcting by using a matrix peak and an enzyme self-cutting peak before testing. Setting instrument parameters: nd: YAG laser, 335nm, 200Hz laser excitation. The original mass spectrum data is input into a matrix science website (http:// www.matrixscience.com) to be searched, and the protein which is theoretically matched with the enzymolysis peptide fragment can be obtained. The main search parameters are: database (Database): NCBIprot; enzyme (Enzyme): trypsin (Trypsin); species (Taxonomy): human (Homo sapiens); fixed modification (Fixed modification): carbamimidomethyl cysteinyl (C); variable modification (variblemodification): oxidation (Oxidation).

FIG. 2 is a two-dimensional electrophoresis pattern of a human serum sample from a control group, which is analyzed by ImageMaster2-D Platinum 5.0 and then an average 376 + -9 protein spots per gel are detected; FIG. 3 is a two-dimensional electrophoresis of human serum samples after interaction with 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound, which detected an average of 356. + -.6 protein spots per gel. Matching the control map with the sample map, using P less than or equal to 0.05 as a standard, and after being analyzed by ImageMaster2-D Platinum 5.0, confirming that the differentially expressed proteins are 20 in total, and as shown in figure 3, the numbers of the differential points are shown in the figure.

Example seven results

20 differentially expressed protein spots were obtained using imagemaster5.0 gel image analysis software and the names of 19 differentially expressed proteins (protein No. 17 with no results identified) were determined by Blast search of NCBInr (see table 2) and are: keratin 1, serum transferrin isoform 1 precursor, complement factor B preprotein, alpha-1-antitrypsin precursor, complement C1S subcomponent subtype 1 preprotein, vitronectin precursor, A1BG protein alpha-1-antitrypsin precursor, kininogen-1 isoform 2 precursor, antithrombin-III precursor, recombinant IgG4 heavy chain, a moiety, haptoglobin isoform 1 preproprotein, alpha-1-antitrypsin precursor, apolipoprotein E isoform B precursor, serum amyloid P component precursor, apolipoprotein a-I subtype 1 preproprotein, apolipoprotein C-III precursor.

TABLE 25 differential protein information after interaction of phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole with human serum samples

EXAMPLE preparation of octa 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole

Dissolving 1-azido-4-methylbenzene (1.0mmol) and styrene (1.0mmol) in a mixed solvent of THF and HMPA (10mL, volume ratio of 1:1) at room temperature, and adding SmI₂(2.0mmol), after stirring for 2 hours (TLC detection of disappearance of starting material), the reaction mixture was concentrated, diluted with ethyl acetate, washed with water and saturated brine in that order, and dried over anhydrous sodium sulfateDrying, filtering, concentrating, subjecting to silica gel column chromatography to obtain 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole (white solid, 221mg, yield 93.9%, mp140-142 deg.C,¹H NMR(CDCl₃,400MHz):δ＝7.86(s,1H),7.42-7.35(m,3H),7.34-7.23(m,6H),2.40(s,3H)；¹³C NMR(CDCl₃,100MHz):δ＝139.3,137.6,134.1,133.3,129.9,129.1,128.8,128.5,126.9,125.0,21.2)。

dissolving 1-azido-4-methylbenzene (1.0mmol) and styrene (1.2mmol) in a mixed solvent of THF and HMPA (10mL, volume ratio of 1:1) at room temperature, and adding SmI₂(2.0mmol), after stirring for 2 hours (TLC detection of disappearance of starting material), the reaction mixture was concentrated, diluted with ethyl acetate, washed with water and saturated brine in this order, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography to give 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole (white solid, 226mg, structure confirmation data in agreement with the report).

Dissolving 1-azido-4-methylbenzene (1.0mmol) and styrene (1.2mmol) in a mixed solvent of THF and HMPA (10mL, volume ratio of 1:1) at room temperature, and adding SmI₂(0.2mmol) and after stirring for 2 hours, TLC detection shows that the reactant in the reaction liquid is still the main point and the target product point is hidden and visible.

Claims

1. A method for detecting differential protein by the action of 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole and serum, which is characterized by comprising the following steps:

(2) removing the high-abundance protein in the sample b to obtain a sample c;

(3) quantifying the protein concentration of sample c by the Bradford method;

(4) performing two-dimensional electrophoresis experiment

(5) film image processing and mass spectrometric identification

(6) results

20 differential expression protein spots are obtained by ImageMaster5.0 gel image analysis software, and the information of No. 1-16 and No. 18-20 differential expression proteins is determined by Blast search of NCBInr,

2. the method according to claim 1, wherein the 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound solution in the step (1) is a methanol solution of 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound, and the concentration of the 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole compound in the sample a is 2.0X 10^-3mol/L。

3. The method according to claim 1, wherein the step (1) comprises incubation at a constant temperature of: the temperature was 37 ℃, the speed r was 280rpm, and the time was 1 h.

4. The method of claim 1, wherein the serum protein is selected from the group consisting of human serum albumin.

5. The method of claim 1, wherein the step (2) of removing the abundant protein is performed according to the AndyBio kit.

6. The method according to claim 1, wherein the gel strip specification in step (4) is 24cm non-linear IPG gel strip IPG pH 3-10 NL.

Use of 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole for determining differential proteins by interaction with serum proteins, characterised in that the 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole is prepared by a process which comprises the steps of:

dissolving 1-azido-4-methylbenzene and styrene in an organic solvent at room temperature, and adding SmI₂Stirring to react for 2 hours to obtain 5-phenyl-1- (p-methylphenyl) -1H-1,2, 3-triazole;