Preparation method and identification method for N-myristic acylation of eimeria tenella
The N-myristoylation of Eimeria tenella was analyzed using a bioorthogonal click chemistry method, which solved the problem of incomplete omics analysis and provided basic data for the development of novel anticoccidial drugs.
Patent Information
- Application Number
- CN202511025749.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-24
- Publication Date
- 2025-11-04
AI Technical Summary
The lack of analytical methods for N-myristoylation of Eimeria tenella hinders the development of novel anticoccidial drugs.
Using bioorthogonal click chemistry, alkynyl-myristoyl acid was used as a probe in combination with copper-catalyzed azidoalkynyl cycloaddition reaction. The target protein was enriched by agarose beads and identified by mass spectrometry to obtain the N-myristoymic map of Eimeria tenella.
This study achieved an effective analysis of the N-myristoylation omics of Eimeria tenella, providing fundamental data for the development of novel anticoccidial drugs.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biochemistry, and particularly relates to a method for preparing and identifying N-myristoylation of Eimeria tenella. BACKGROUND
[0002] Myristoylation is a 14-carbon saturated fatty acid, myristic acid, attached to the N-terminal glycine of a subset of eukaryotic proteins. Although often classified as a post-translational modification (PTM), it usually occurs during the co-translational phase. Specifically, when the ribosome has polymerized less than 100 residues, and after the removal of the N-terminal methionine by methionine aminopeptidase, the N-terminal glycine is exposed. The enzyme that catalyzes the transfer of the myristoyl group is myristoyl CoA:N-myristoyl transferase (NMT). The myristoyl CoA substrate first binds to the apoprotein, causing a conformational change that allows the peptide substrate to bind, the N-terminal glycine amine to attack the myristoyl CoA (myr-COA) thioester, and the chemical transformation to occur by nucleophilic substitution; the COA product is released first, followed by the myristoylated peptide.
[0003] Specifically, myristoylation is involved in a variety of biological functions, including signal transduction, cellular localization, and tumorigenesis. There are also studies showing that protein N-myristoylation is involved in the host's defense against microbial and viral infections.
[0004] N-myristoylation occurs co-translationally in eukaryotes, involving the attachment of myristic acid to the N-terminal glycine of a variety of substrate proteins. Although N-myristoylation usually occurs on newly synthesized polypeptides after the removal of methionine by methionine aminopeptidase (MetAP2), there is also evidence that post-translational myristoylation occurs on internal glycines exposed by caspase cleavage during apoptosis. Myristoylation plays an important role in signal transduction, protein stability, and protein localization on membranes.
[0005] One of the key chemical reactions employed therein is the azide alkyne click chemistry reaction. The azide alkyne Click reaction is a highly efficient chemical reaction, which is applied in the fields of organic synthesis and medicinal chemistry due to its high selectivity and high yield and mild reaction conditions, etc., especially the copper-catalyzed azide alkyne cycloaddition reaction (CuAAC).
[0006] It mainly includes three processes: 1. Copper ion catalysis: under the catalysis of copper ions, the azide group and the alkyne group first form copper-alkyne complex and azide-copper complex intermediates; 2. Cycloaddition reaction: the above intermediates undergo cycloaddition reaction to generate 1,2,3-triazole ring structure; 3. Protonation: finally, the triazole-copper derivative is protonated to obtain the triazole product, completing the catalytic cycle.
[0007] The analysis of Eimeria tenella N-myristoylome is the basis for the development of new anticoccidial drugs. However, unfortunately, there is no report on the N-myristoylome data of Eimeria tenella. SUMMARY
[0008] Therefore, the present application aims to provide a method for analyzing the N-myristoylome of Eimeria tenella by using bio-orthogonal click chemistry, so as to solve the problem of imperfect analysis mechanism of Eimeria tenella N-myristoylome.
[0009] The present application provides a preparation method of Eimeria tenella N-myristoylome, which comprises the following steps:
[0010] (1) taking the second generation of merozoites;
[0011] (2) adding alkynyl-myristoyl acid as a probe into the cell culture medium to culture the second generation of merozoites, and lysing the cells to obtain a lysate after the culture is completed;
[0012] (3) performing copper-catalyzed azide alkyne cycloaddition reaction on the lysate, adding N-ethylmaleimide to terminate the reaction and incubating overnight at low temperature to obtain a reaction product;
[0013] (4) performing precipitation operation on the reaction product, and using agarose beads to enrich the target protein;
[0014] (5) using an eluent to wash the agarose beads to obtain the target protein.
[0015] In the present application, the working concentration of alkynyl-myristoyl acid in step (2) is 50 μM.
[0016] In the present application, the reaction system in step (3) comprises:
[0017] CUAAC click labeling reagent, diazobiotin-triglycol-azide, copper sulfate, tris(2-carboxyethyl) phosphine and tert-butyl trichloroacetimidate.
[0018] In the present application, the component concentration of the reaction system in step (3) is:
[0019] The concentration of diazobiotin-triglycol-azide is 10-20 mM; the concentration of copper sulfate is 40-60 mM; the concentration of tris(2-carboxyethyl) phosphine is 40-60 mM; and the concentration of tert-butyl trichloroacetimidate is 2-4 mM.
[0020] In the present application, the added concentration of N-ethylmaleimide in step (3) is 0.4-0.6 M.
[0021] In the present application, the precipitation operation in step (4) is specifically as follows:
[0022] The pre-cooled methanol and chloroform are added for precipitation operation, and the operation is repeated three times.
[0023] The volume ratio of the methanol to the chloroform is 600:150.
[0024] In the present application, the agarose beads in step (4) are streptavidin-modified agarose beads.
[0025] The present application also provides a method for identifying N-myristoylation of Eimeria tenella, which comprises the following steps: performing mass spectrometry identification on a target protein to obtain a myristoylome profile.
[0026] The mass spectrometry identification adopts liquid chromatography-tandem mass spectrometry, and the chromatographic column in the liquid chromatography-tandem mass spectrometry is a C18 reversed-phase column, the particle size of the C18 reversed-phase column is 1.8 μm, the inner diameter is 0.15 mm, and the length is 100 mm.
[0027] In the present application, after obtaining the data of mass spectrometry identification, the identification method is used to search the database by comparing a database specific to Eimeria tenella through MaxQuant 2.0 software.
[0028] The search parameters include: enzyme cutting mode: trypsin complete enzyme cutting; dynamic modification: myristoylation modification; peptide mass tolerance: 20 ppm; fragment ion tolerance: 0.05 Da.
[0029] In the present application, the second generation merozoite of Eimeria tenella is used, alkynyl-myristic acid is used as a myristoyl probe, the second generation merozoite is cultured in a cell culture medium for a period of time, then washing and lysis are performed, finally, a lysis solution is added to lyse the cells, and ultrasonic crushing is performed, then a copper-catalyzed azide alkyne cycloaddition reaction is performed, a CUAAC click labeling reagent is added for labeling, then diazo biotin-PEG3-azide, copper sulfate, tris(2-carboxyethyl) phosphine (TCEP) and tert-butyl trichloroacetimidate (TBTA) are added, incubated for a period of time, then N-ethylmaleimide (NEM) is added, then low-temperature incubation is performed overnight, then methanol and chloroform precipitation operation is performed three times, then agarose beads are used for protein enrichment, the target protein is obtained by washing the beads with an eluent, the obtained protein is the substrate of myristoylase, that is, the total protein modified by myristoylation, then mass spectrometry identification is performed to obtain a myristoylome profile. BRIEF DESCRIPTION OF DRAWINGS
[0030] Figure 1 It is a mass spectrometry identification profile.
[0031] Figure 2 for mass spectrometry identification map;
[0032] Figure 3 for search results. DETAILED DESCRIPTION
[0033] Hereinafter, when a specific technique or condition is not mentioned for the reagent or instrument used, it is performed according to the conventional experimental condition, and when the reagent company instruction is not explicitly mentioned, it is performed according to the condition suggested in the instruction. When the manufacturer of the reagent or instrument used is not mentioned, it is a conventional product that can be obtained by purchase on the market.
[0034] EMBODIMENT
[0035] I. Analysis of N-myristoylation of Eimeria tenella by using bio-orthogonal click chemistry
[0036] In this study, the second generation of Eimeria tenella merozoites was used, and alkynyl-myristic acid was used as a myristoyl probe into the cell culture medium. After culturing the second generation of merozoites for a period of time (at least 12 h), followed by washing and lysis, finally adding lysis solution to lyse the cells, and ultrasonic crushing, followed by copper-catalyzed azide alkyne cycloaddition reaction, adding CUAAC click labeling reagent for labeling, followed by adding diazo biotin-PEG3-azide, copper sulfate, tris(2-carboxyethyl) phosphine (TCEP) and tert-butyl trichloroacetimidate (TBTA), incubating for a period of time, then adding N-ethylmaleimide (NEM), followed by low-temperature incubation overnight, followed by three times of methanol and chloroform precipitation operation, followed by protein enrichment using agarose beads, and the target protein was obtained by washing the beads with eluent. The obtained protein is the substrate of myristoylase, that is, the myristoylated total protein, followed by mass spectrometry identification, and the myristoylation group spectrum is obtained. Specifically, the implementation process of a method for analyzing N-myristoylation of Eimeria tenella by using bio-orthogonal click chemistry includes:
[0037] 1. Preparation of the second generation of Eimeria tenella merozoites
[0038] Eimeria tenella was used to attack and infect 14-day-old chicks, and the cecum was collected 5 days after the attack and infection of the oocysts, and hyaluronidase digestion was used to obtain the second generation of Eimeria tenella merozoites. After washing with PBS, it was added to the cell culture medium and incubated in a carbon dioxide incubator.
[0039] 2. N-myristoylated protein enrichment
[0040] (1) Add the appropriate amount of alkynyl-myristoyl acid (12 μL of 0.05M alkynyl-myristoyl acid) to the cell culture medium containing the second generation of schizonts (probe working concentration of 50 μM), and incubate the cells for 12 hours. After washing the cells with PBS, centrifuge to obtain the cell precipitate.
[0041] (2) Add the cell precipitate obtained in step 1 to 190 μl of lysis solution, and add 10 μl of 0.1M PMSF to lyse the cells. Sonicate at a power of 300W for 5 seconds, stop for 5 seconds, and repeat for 2 minutes. Then centrifuge at 12000g for 5 minutes, and take the supernatant. Measure the protein concentration using the BCA method, and take 500 μg of the protein solution into a 0.5ml EP tube. Add PBS to make up to 180 μl.
[0042] (3) Add 20 μl of CUAAC click labeling reagent for labeling. The solution contains diazo biotin-PEG3-azide (10-20mM); copper sulfate (40-60mM); tris (2-carboxyethyl) phosphine (TCEP) (40-60mM); t-butyl trichloroacetimidate (TBTA) (2-4mM). Then add 15 μl of N-ethylmaleimide (NEM) (0.4-0.6M) dissolved in DMSO, and incubate at room temperature for 4 hours (or incubate overnight at 4°C. Before overnight incubation, it is recommended to add 2 μl of 0.1M PMSF).
[0043] (4) Add pre-cooled 600 μl of methanol, 150 μl of chloroform, and 400 μl of deionized water, mix well, and centrifuge at 6000rpm for 15 minutes at 4°C. After centrifugation, a clear white film is visible in the middle of the solution. Absorb the liquid above and below the film and retain the film. Add pre-cooled 600 μl of methanol, centrifuge at 6000rpm for 5 minutes at 4°C. After centrifugation, remove the upper layer of methanol. After natural air-drying of the precipitate for 2-3 minutes, add 200 μl of lysis solution to shake and mix the dissolved precipitate. Repeat the previous step, and mix the precipitate with 200 μl of lysis solution. Keep ready for use.
[0044] (5) Take 60 μl of streptavidin agarose beads into a 0.5ml EP tube, add 500 μl of PBS to wash the agarose beads, invert to mix well for 15 times, centrifuge at 2000g for 2 minutes, remove the supernatant, and repeat twice.
[0045] (6) After washing, add 50-70 μl of elution solution to the agarose gel beads to elute the target protein, and incubate at 37°C for 15 minutes. Centrifuge at room temperature at 15000g for 1 minute, collect the supernatant, and store at -80°C.
[0046] II. Mass spectrometry identification
[0047] 1. Proteolysis
[0048] (1) Add appropriate trypsin to the target protein prepared, and incubate overnight at 37°C in a constant temperature shaker.
[0049] (2) After proteolysis, store the peptide solution at -80°C, and desalt.
[0050] 2. Desalting and lyophilization
[0051] (1) Desalt the polypeptide sample using C18 tips, then pre-equilibrate with 50% ACN, then wash with 0.1% trifluoroacetic acid, slowly and repeatedly blow and suck the C18 tips in the sample dissolved in 0.1% trifluoroacetic acid to make the C18 tips fully combined with the sample, after combination, wash with 0.1% trifluoroacetic acid / 5% ACN, and finally elute the polypeptide with 100 μl of a solution containing 50% acetonitrile and 0.1% trifluoroacetic acid.
[0052] (2) Vacuum concentrate and spin dry the sample after desalting purification.
[0053] (3) Then use liquid chromatography tandem mass spectrometry to determine the mass spectrum.
[0054] Reverse column information: C18 column (1.8 μm, 0.15 x 1,00 mm)
[0055] Chromatographic instrument: Thermo Fisher Easy-nLC 1000
[0056] Mass spectrometer: Thermo Fisher Q Exactive HF-X
[0057] Chromatographic separation time: 98 minutes
[0058] Solution A: 0.1% formic acid aqueous solution
[0059] Solution B: 100% acetonitrile solution
[0060] Flow rate: 600 nL / min
[0061] Gradient: as shown in Table 1.
[0062] Table 1 Gradient
[0063] Time (min) A％ B％ Flow rate (nL / min) 0 96 4 300 2 93 7 300 80 75 25 300 84 65 35 300 86 10 90 600 94 10 90 600 96 96 4 300 102 96 4 300
[0064] 3. Mass spectrometry identification parameters
[0065] Table 2 Mass spectrometry identification parameters
[0066]
[0067]
[0068] By data alignment, QMREVVELPLTNPELFK and HYFSNTHGIIFVVDSNDR are shown to belong to 26S proteasome regulatory ATPase subunit, putative (Uniprot No. U6KLG7) and ADP-ribosylation factor (Uniprot No. U6KXG0), respectively. For details, refer to Figure 1 and Figure 2 .
[0069] 4. Data analysis
[0070] Mass spectrometry raw data: The raw mass spectrometry data is a RAW format file, and Maxquant 2.0 (Thermo) is used for library searching identification and analysis.
[0071] Database: The data used in this case is: uniprot-Eimeria tenella.Fasta
[0072] Library searching setting:
[0073] Library searching uses Maxquant2.1 (Thermo), and the related parameters are shown in Table 3.
[0074] Table 3 Library searching parameters
[0075] Input Data Enzyme Name Trypsin (Full) Max. Missed Cleavage Sites 2 Static Modification Acetyl [N-Term] Oxidation Dynamic Modification (myristoylation) Precursor Mass Tolerance 20 ppm Fragment Mass Tolerance 0.05D Validation based on q-Value
[0076] The library searching result is shown in Figure 3 .
[0077] It can be known from the above detection that the N-myristoylation of Eimeria tenella is resolved by using the biological orthogonal click chemistry method.
[0078] The above description is only the preferred embodiment of the present application, and cannot be used to limit the implementation range of the present application. Changes and improvements of the present application should be included in the protection range of the present application.
Claims
1. A method for preparing N-myristylated Eimeria tenella, characterized in that, The preparation method includes the following steps: (1) Obtain second-generation merozoites; (2) The second generation merozoites were cultured by adding alkynyl-myristoyl acid as a probe into the cell culture medium. After the culture was completed, the cells were lysed to obtain the lysate. (3) The pyrolysis solution was subjected to a copper-catalyzed cycloaddition reaction of azide ytylene, and N-ethylmaleimide was added to terminate the reaction and the mixture was incubated at low temperature overnight to obtain the reaction product; (4) The reaction product is precipitated and the target protein is enriched using agarose beads; (5) Wash the agarose beads with elution buffer to obtain the target protein.
2. The preparation method according to claim 1, characterized in that, The working concentration of alkynyl-myristoyl acid in step (2) is 50 μM.
3. The preparation method according to claim 1, characterized in that, The reaction system in step (3) comprises: CUAAC click labeling reagent, diazobiotin-polyethylene glycol-azide, copper sulfate, tris(2-carboxyethyl)phosphine and tert-butyltrichloroacetylimine ester.
4. The method according to claim 1, characterized in that, The component concentrations of the reaction system in step (3) are: Diazobiotin-polyethylene glycol-azide at 10–20 mM; copper sulfate at 40–60 mM; tris(2-carboxyethyl)phosphine at 40–60 mM; tert-butyltrichloroacetylimine at 2–4 mM.
5. The method according to claim 1, characterized in that, The concentration of N-ethylmaleimide added in step (3) is 0.4-0.6M.
6. The method according to claim 1, characterized in that, The precipitation operation in step (4) is specifically as follows: Pre-cooled methanol and chloroform were added to induce precipitation, and this process was repeated three times. The volume ratio of methanol to chloroform is 600:
150.
7. The method according to claim 1, characterized in that, The agarose beads in step (4) are streptavidin-modified agarose beads.
8. A method for identifying N-myristylation of Eimeria tenella, characterized in that, The identification method includes the following steps: mass spectrometry identification of the target protein to obtain a cardamomycium mics map; The mass spectrometry identification was performed using liquid chromatography-tandem mass spectrometry, with a C18 reversed-phase column. The C18 reversed-phase column has a particle size of 1.8 μm, an inner diameter of 0.15 mm, and a length of 100 mm.
9. The identification method according to claim 8, characterized in that, After obtaining the mass spectrometry identification data, the identification method uses MaxQuant 2.0 software to perform a database search analysis against a dedicated database of Eimeria tenella. The search parameters include: Enzymatic digestion method: complete digestion with trypsin; Dynamic modification: cardamomylation modification; Peptide mass tolerance: 20 ppm; Fragment ion tolerance: 0.05 Da.