CN112630446A - Bioactive molecule combined target identification method based on double-head photoaffinity probe - Google Patents
Bioactive molecule combined target identification method based on double-head photoaffinity probe Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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Abstract
The invention relates to the technical field of medicines, and provides a method for identifying a bioactive molecule combined target based on a double-headed photoaffinity probe. The double-head photoaffinity probe is used for extracting a protein compound from whole cells, the target is specifically combined through ultraviolet light capture and polyacrylamide gel electrophoresis separation technology, the non-specificity of target identification is greatly reduced, the method can be used for identifying and researching the combined target of bioactive small molecules, is beneficial to finding brand new disease combined targets, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of medicines, relates to a method for identifying a bioactive molecule combined target, and particularly relates to a method for identifying a bioactive molecule combined target based on a double-ended photoaffinity probe.
Background
The light affinity labeling technology (PAL) combines the advantages of modern molecular biology, cell biology, medicinal chemistry, analytical chemistry and other subjects, and applies the synthesized light affinity probe molecules to generate high-activity intermediates under the irradiation of light with specific wavelength, which can be directly irreversibly covalently crosslinked with the protein specifically combined with the medicinal molecules to realize the capture of the medicinal target protein molecules. By using the light affinity labeling technology, a large number of targets related to diseases are found, the mode of action of a plurality of small molecules and biological macromolecules is determined, and the information has very important significance on rational drug design, such as discovery of new drug targets or new description of the function of a known protein, which is self-evident in the significance of clarifying the occurrence, development and treatment of diseases from the molecular level.
Photoaffinity labeling probe design plays a crucial role in the implementation of Photoaffinity labeling technology, but when a part of biologically active small molecules is analyzed by using a classical connecting skeleton (such as lysine, polyethylene glycol, alkyl chain skeleton, etc.), the probe has limited sensitivity due to the branched structure skeleton of the whole probe, and non-specific binding exists, so that the target cannot be completely and accurately identified (Salisbury, C.M. and B.F. Cravatt (2008) 'Optimization activity-based probes for structural purification of tissue enzymes complex.' J Am Chem Soc 130(7): 2184-.
In order to solve the above technical problems, the inventors developed and improved the existing probes, (Wang, d.y., y.cao, l.y.zheng, l.d.chen, x.f.chen, z.y.hong, z.y.zhu, x.li and y.f.chai (2017). "Target Identification of Kinase Inhibitor alert (MLN8237) by Using DNA-Programmed Affinity labelling" Chemistry 23(45):10906 and 10914.) to provide a photoaffinity probe set consisting of a binding probe and a capture probe, which are base complementary paired or specifically bound between the two probes, one side of the binding probe is bound to a bioactive molecule, and one side of the capture probe is bound to a photoaffinity molecule. When the target identification is carried out, the combined probe and the capture probe are respectively hybridized, the probe with a certain concentration and a sample are incubated together, and the incubated sample is placed under the excitation light source of the photoaffinity molecule for irradiation, and the cross-linking reaction is excited, so that the capture probe can effectively anchor the relevant target protein to form a probe-protein composite.
Compared with the current probe molecules, the single-head probe has the following advantages because the structure skeleton is concentrated and is a flexible structure: the sensitivity is obviously increased, and the recognition capability of the target protein pair with low abundance and low affinity is improved. However, cells are filled with hydrophobic interaction and electrostatic interaction, and thousands of proteins with different biophysical characteristics and abundances, so weak or transient nonspecific interaction generally exists, and with the development of proteomics and protein mass spectrometry identification technologies, the number of identified potential bioactive small molecule binding proteins is remarkably increased, and the difficulty of one-by-one verification is high and the efficiency is low. Therefore, how to optimize the single-head probe target analysis method reduces the non-specificity while ensuring the sensitivity, and has important significance for small molecule target analysis.
Disclosure of Invention
The invention aims to research a double-head photoaffinity probe aiming at the defects of a single-head probe, and provides a method for identifying a bioactive molecule combined target based on the double-head photoaffinity probe, which comprises the following specific steps:
the invention provides a method for identifying a bioactive molecule combined target based on a double-head photoaffinity probe, which comprises the following steps:
s1 preparation of double-ended photoaffinity probe
Taking groups as units, wherein each group consists of a double-head binding probe and a double-head capture probe; complementary pairing or specific combination of the probe framework of the double-head binding probe and the base of the probe framework of the double-head capture probe; the two sides of the probe framework of the double-head binding probe are combined with the same bioactive molecules, and the two sides of the probe framework of the double-head capture probe are combined with the same photoaffinity molecules.
Two ends of the probe skeleton are subjected to amino modification and then react with activated ester of bioactive molecules or photoaffinity molecules to respectively obtain a double-headed binding probe and a double-headed capture probe, and the double-headed binding probe and the double-headed capture probe are purified and characterized for later use;
s2 protein sample preparation
Extracting a protein sample from a biological sample, and enriching after testing content and purity;
s3, capture, separation and identification of bioactive small molecule binding target
3.1 capture:
respectively hybridizing the double-headed binding probe and the double-headed capture probe, incubating the double-headed photoaffinity probe with a protein sample at a certain concentration, irradiating the double-headed photoaffinity probe and the protein sample under a photoaffinity molecule excitation light source, exciting a light cross-linking reaction to ensure that the capture probe is effectively anchored with a related target protein, and specifically binding the double-headed binding probe and the double-headed capture probe to form a protein-probe-protein covalent complex;
3.2, extraction:
primarily removing unbound free protein from the mixed sample, and drying and dissolving the obtained precipitate; after purifying and enriching the protein-probe-protein complex, removing free probes;
3.3 isolation and identification:
and (3) after the sample in the step (3.2) is dissolved, separating the bioactive small molecule combined target by a certain method, and identifying the target to obtain the bioactive molecule combined target.
Preferably, in S1, the probe scaffold is a DNA scaffold, an RNA scaffold or a polypeptide scaffold; the biological molecules comprise small molecules, aptamers, polysaccharides, polypeptides or proteins; the photoaffinity molecule comprises aromatic azide, diazomethane or benzophenone; the double-ended capture probe is connected with a biological reaction amplification system, and the biological reaction amplification system can select a common biotinylated base T which is synthesized by a Click reaction.
Preferably, in S1 the first step of the method,
the double-headed binding probe was prepared as follows: firstly, preparing carboxyl or sulfo derivatives of bioactive small molecules, and preparing activated esters of the derivatives by EDC/NHS coupling reaction; secondly, amino modification is carried out at two ends of the probe skeleton, and the double-end combined probe is synthesized by further utilizing the condensation reaction of the amino and activated ester.
The preparation process of the double-head capture probe is as follows: firstly, preparing carboxyl or sulfo derivatives of photoaffinity molecules, and preparing activated esters of the derivatives by EDC/NHS coupling reaction; secondly, amino modification is carried out at two ends of the probe skeleton, and the double-end capture probe is synthesized by further utilizing the condensation reaction of the amino and activated ester.
The purification of the double-ended light affinity probe is carried out by adopting the steps of combining ethanol precipitation and chromatographic separation:
ethanol precipitation: measuring the volume of the mixed solution, firstly adding sodium acetate with pH of 5.0 and concentration of 3mol/L in one tenth volume, then adding glycogen with 7.5mg/mL in one tenth volume, then adding absolute ethyl alcohol with 2-3 times volume, uniformly mixing, placing at-80 ℃, and placing for at least 2 hours; taking out a sample, centrifuging 17000g at 4 ℃, discarding the supernatant, adding 70% precooled ethanol into the precipitate, washing by shaking, and centrifuging 17000g at 4 ℃; drying the obtained precipitate, dissolving with triethylamine acetate buffer solution, purifying by preparative chromatography,
and (3) preparing a chromatogram: adopting preparative high performance liquid chromatography; mobile phase: phase A: 0.1mol/L triethylamine acetate buffer solution; phase B: acetonitrile; a chromatographic column: agilent ZORBAX XDB C18 chromatography column; detection wavelength: 210-400 nm; after elution, vacuum freeze-drying is carried out, ultra-pure water is used for redissolving, and then the concentration of the product is measured by an ultra-micro fluorescence spectrophotometer,
and (4) carrying out purity and molecular weight determination characterization on the purified probe for later use.
Preferably, in S2, the protein sample is derived from cultured cells, animal tissue or human specimens. The protein sample extraction method comprises lysate extraction, repeated freeze thawing method or ultrasonic extraction method.
Preferably, in S3, the method for separating the bioactive small molecule bound target comprises electrophoretic separation or high performance liquid chromatography separation; identification of the bioactive small molecule binding target comprises an immunoblotting technique or a protein mass spectrometry identification technique; the verification of the combination of the bioactive small molecule and the target comprises a surface plasma resonance test, an isothermal titration calorimetry test, a cell thermal transition analysis test or a drug affinity target stability test.
The invention has the following beneficial guarantee and effects:
through experiments, the double-end photoaffinity probe prepared by the method has high purity, and the actually measured molecular weight is close to the theoretical molecular weight, so that the actual detection requirement is met.
The invention discovers that the double-head photoaffinity probe is used for extracting a protein compound from whole cells, the nonspecific identification of the target is greatly reduced by ultraviolet light capture and polyacrylamide gel electrophoresis separation technology visible specific binding target, the double-head photoaffinity probe can be used for identifying and researching the binding target of bioactive small molecules, is beneficial to discovering brand new disease binding targets, and has good application prospect.
Through verification by target identification of the small biomolecule MLN8237, the method can effectively identify the specific binding target and the non-specific binding target of the MLN 8237; compared with a single-head photoaffinity probe, the double-head photoaffinity probe disclosed by the invention has better sensitivity and accuracy in target identification.
Drawings
FIG. 1 shows the characterization results of the double-ended DNA photoaffinity probe: a is a double-head binding probe mass spectrum identification result, B is a double-head binding probe purity detection result, C is a double-head capture probe mass spectrum identification result, and D is a double-head capture probe purity detection result.
FIG. 2 is a comparison of the capture of aurora A, tubulin, HSP60 in cell whole protein extract by MLN8237 single-headed and double-headed DNA photoaffinity probes: a is a schematic diagram of two probe forms, B is a single-head and double-head DNA light affinity probe with the length of 16bp base, and C is a single-head and double-head DNA light affinity probe with the length of 100bp base.
FIG. 3 shows the target validation results of MLN8237, wherein A is the Tubulin binding surface plasmon resonance identification result and B is the HSP60 binding surface plasmon resonance identification result.
Detailed Description
The present invention will now be described in detail with reference to examples and drawings, but the practice of the invention is not limited thereto.
The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Percentages and parts are by volume unless otherwise indicated.
The identification process of the present invention will be described below by taking the target identification process of the small biological molecule MLN8237 with relatively clear target as an example.
Example 1: double-ended photoaffinity probe preparation, purification and characterization
1. Preparation of double-ended photoaffinity probe
The double-head light affinity probe takes a group as a unit and consists of a double-head binding probe and a double-head capture probe together. The double-headed binding probes are similar to the double-headed capture probes in structure, and each comprises a probe skeleton in the middle, and active molecules and a biological reaction amplification system bound on two sides of the skeleton, as shown in FIG. 2A.
The two sides of the probe framework of the double-head binding probe are combined with the same bioactive molecules, the two sides of the probe framework of the double-head capture probe are combined with the same photoaffinity molecules, and the probe framework of the double-head binding probe is complementarily paired with or specifically combined with the base group of the probe framework of the double-head capture probe.
In this embodiment, the probe skeleton includes, but is not limited to, a DNA skeleton, an RNA skeleton, a polypeptide skeleton, etc.; biomolecules include, but are not limited to, small molecules, aptamers, polysaccharides, polypeptides, proteins, and the like.
(1) Preparation of double-headed binding probes:
firstly, preparing carboxyl or sulfo derivatives of bioactive small molecules MLN8237, and preparing activated esters of the derivatives by EDC/NHS coupling reaction; secondly, amino modification is carried out on the 5 ' end and the 3 ' end of the DNA binding chain, wherein 5 ' amino TFA (CAS number: 133975-85-6, English name 5 ' -amino-modifier-C6-TFACEP) is used at the 5 ' end, C7 amino solid phase carrier (CB number: CB8356712, English name 3 ' -DMT-TC6(NH-FMOC) -CPG) is directly used at the 3 ' end, and the DNA binding chain is synthesized according to the conventional primers; and further utilizing the condensation reaction of amino and activated ester to synthesize the double-end combined probe.
The synthetic route of the binding probe of the bioactive small molecule MLN8237 is as follows:
(2) preparing a double-head capture probe:
first, biotinylated base T was synthesized by the Click reaction as a biological reaction amplification system.
Secondly, carrying out amino modification on the 5 'end and the 3' end of the DNA capture chain, then preparing 4-azidobenzoic acid activated ester by EDC/NHS coupling reaction, and further carrying out synthesis by utilizing condensation reaction of amino and activated ester.
The synthetic route of the capture probe is as follows:
in practical synthesis applications, capturing of the bioactive small molecule binding target includes but is not limited to the above-mentioned phenyl azide ultraviolet free radical crosslinking capture, including but not limited to aromatic azides, diazomethanes, benzophenones, and the like.
(3) And (3) purification of the probe: ethanol precipitation followed by preparative chromatography
Ethanol precipitation: measuring the volume of the mixed solution, adding 3moL/L sodium acetate (pH5.0) of one tenth volume, adding 7.5mg/mL glycogen of one tenth volume, adding 2.5 times volume of absolute ethyl alcohol, mixing uniformly, placing at-80 ℃, and placing for at least 2 h; taking out the sample, centrifuging at 17000g for 15min at 4 ℃, discarding the supernatant, adding 70% precooled ethanol into the precipitate, washing with shaking, and centrifuging at 17000g for 5min at 4 ℃; and drying the obtained precipitate, dissolving the precipitate by using triethylamine acetate buffer solution, and performing preparative chromatographic purification treatment.
And (3) preparing a chromatogram: the system comprises the following steps: agilent 1200 preparative high performance liquid chromatography. Mobile phase: phase A: 0.1moL/L triethylamine acetate buffer (Triethylamineacetate, TEAA); phase B: and (3) acetonitrile. A chromatographic column: agilent ZORBAX XDB C18 column (4.6 mm. times.250 mm, 5 μm). A detector: a multi-wavelength ultraviolet detector (210nm to 400 nm). After elution, vacuum freeze-drying was performed, and then reconstituted with ultrapure water, and the concentration was measured with an ultramicro fluorescence spectrophotometer.
(4) Characterization of the probes:
and (3) purity determination: the purity of the probe is measured by adopting a high performance liquid chromatography technology, 0.1 percent of formic acid water and 0.1 percent of formic acid acetonitrile are used as mobile phases, the large gradient elution is carried out for 0-15min and the organic phase is 5-95 percent, the ultraviolet full-wavelength detection is carried out, the purity of the probe is calculated according to the chromatographic peak area in a chromatogram, and the purity is more than 95 percent.
And (3) measuring the molecular weight: and (2) determining the molecular weight distribution of the probe by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometer for characterization and identification, wherein the matrix is A: b ═ 8: 1, wherein A is 50mg/mL of 3-hydroxypropanal acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 1: 1), and B is 50mg/mL of ammonium citrate aqueous solution.
In this example, an MLN 8237-double-ended DNA photoaffinity probe with a base number of 100bp was synthesized by amino-carboxyl coupling reaction, and further molecular weight measurement was performed by mass spectrometry and purity detection was performed by reverse phase chromatography. Information regarding binding probes and capture probes in MLN8237 photoaffinity probes is summarized in Table 1:
TABLE 1 MLN8237 photoaffinity probe information
The mass spectrum identification result of the binding probe is shown in figure 1A, the actually measured molecular weight of the binding probe is 32645.10, which is extremely close to the theoretical molecular weight, the purity detection result is shown in figure 1B, and the purity of the probe is calculated according to the chromatographic peak area in the chromatogram, and the purity is 97.3%; the mass spectrum identification result of the capture probe is shown in figure 1C, the actually measured molecular weight is 31444.26, the purity detection result is shown in figure 1D, the purity is 98.2%, and the requirements of being more than 95% are met.
Example 2 target identification and validation
1. Extraction of Whole cell protein samples
(1) Cell culture and pretreatment:
human hepatoma cell Hep3B was thermostatted at 37 ℃ with 5% CO2Culturing in cell culture box in DMEM high sugar medium containing 10% fetal calf serum, 0.1mg/mL streptomycin, and 100U/mL penicillin, digesting with 0.25% pancreatin when growth is about 80%, adding appropriate amount of serum-containing medium to stop digestion, centrifuging for 3min at 1000g, discardingThe clear solution was resuspended in 1000g of PBS for 3min and washed once.
(2) Protein extraction:
resuspending the cells with western and IP cell lysate, standing in liquid nitrogen for 3min, standing in 37 deg.C water bath for 5min, repeating for 3 times, centrifuging at 12000g for 5min, and collecting supernatant. Trypan blue staining was observed and compared under a microscope before and after protein extraction.
(3) And (3) content and purity determination:
content determination: protein concentration determination a commercial BCA concentration determination kit was used, following the instructions. Adding the diluted standard substance and the sample to be detected into a 96-well plate, adding an equivalent BCA reaction reagent, incubating for 20min at 37 ℃, reading the absorbance value of the 96-well plate by an enzyme-labeling instrument at a wavelength of 562nm, and calculating according to a standard curve.
And (3) purity determination: adding sodium acetate, glycogen and absolute ethyl alcohol into equivalent protein according to the method for ethanol precipitation, centrifuging to obtain nucleic acid precipitate, adding a proper amount of ultrapure water for dissolving, measuring the concentration of the nucleic acid precipitate by using Thermo nanodrop, calculating and comparing the content of the nucleic acid, further balancing for 2min at room temperature by using a commercial DEAE adsorption column, centrifuging for 5min at 12000g, and removing DNA impurities.
Protein enrichment: using an ultrafiltration tube with 0.3KDa cut-off parameter, centrifuging at 12000g for 2min, and eluting.
2. Capture, isolation and identification of bioactive small molecule binding targets
(1) Capturing:
and hybridizing the binding probe and the capture probe respectively, and incubating the double-ended DNA photoaffinity probe with a protein sample at the temperature of 4 ℃ in a shaking table. The probe is placed under an ultraviolet point light source for irradiation, the irradiation wavelength is 365nm, the irradiation temperature is 4 ℃, and the cross-linking reaction is excited to ensure that the probe is effectively anchored with the related target protein to form a protein-DNA-protein covalent complex.
(2) Extraction:
primarily removing other unbound free protein from the mixed sample by using an ethanol precipitation method, drying the obtained precipitate, and dissolving the dried precipitate by using a phosphate buffer solution or ultrapure water; purifying and enriching a protein-DNA-protein compound by using a biotin-streptavidin magnetic bead method, and optimizing an elution method; free probes were removed by ultrafiltration and the resulting liquid was lyophilized in vacuo for subsequent studies.
(3) Separation and identification:
the sample was added with an appropriate amount of ultrapure water, and 5 Xprotein loading buffer was added thereto in an amount of 1/4, followed by boiling. The method is carried out by adopting a polyacrylamide gel electrophoresis method, and the experimental steps are detailed in the related documents in the prior art through the steps of separation, membrane transfer, antibody incubation, scanning imaging and the like.
3. Validation of biologically active small molecule binding targets
The surface plasmon resonance method verifies the combination between the bioactive small molecule and the combined target: using a BiacoreTMT200 apparatus (GE Health Co.).
Protein coupling: the protein was coupled to CM5 chips by amino coupling using PBS as working buffer and the coupled protein was diluted to a final concentration of 200. mu.g/mL with 10mM NaAc. The chip surface was treated with 0.2M EDC and 50mM NHS at 1:1 mixing for 7 minutes at a flow rate of 10. mu.L/min, protein solution was injected for 7 minutes, and the activated chip surface was blocked by 1M ethanolamine injection for 7 minutes at pH 8.5.
Primary screening: samples were diluted with PBS to 500. mu.M compound with 5% DMSO and then 50. mu.M compound. The sample is injected for 30s at a flow rate of 30 mu L/min, and the binding state of the compound and the protein is observed.
And (3) dynamic experiments: 50 μ M high concentration compounds containing 5% DMSO were prepared, and then diluted 2-fold to give a series of concentration gradients, and the working buffer was 5% DMSO in PBS. Samples were injected at a flow rate of 30. mu.L/min for 60s or 120s, kinetic experiments were performed by dissociation for 300s, and the obtained data were fit analyzed using a kinetic model or a stable binding model in Biacore T200 evaluation software. The larger the signal response value, the lower the Kd value, indicating a stronger physical binding between the active component and its target protein.
4. Results of the experiment
4.1 target identification results
MLN8237 is also called alisertib, and is a clinical candidate drug because it specifically inhibits aurora kinase A (aurora Kinasea), both of which bind to an ATP-binding domain common to kinase-like proteins, and thus MLN8237 can theoretically bind to other various kinases or proteins. The former single-head old method was used to identify the binding target of MLN8237, and Aurora Kinase A (Aurora A) belongs to specific binding, and alpha-tubulin 8(tubulin) and Heat shockprotein (HSP60) belong to non-specific binding.
Comparison of MLN8237 Single-headed and double-headed DNA photoaffinity probes for capture of aurora A, tubulin, HSP60 in cell whole protein extract: wherein the working concentration of the probe is 0.1 mu M, the total cell protein is 500mg, the incubation is carried out for 2h, the ultraviolet light is irradiated for 30s, after the light cross-linking, the protein loading buffer solution is added, the metal bath is carried out for 10min at the temperature of 95 ℃, and the three proteins are detected by the immunoblotting method.
As shown in FIG. 2, in the 16bp sequence, the present double-headed method is not different from the original single-headed method, and the captured sample only contains "aurora A-DNA", "tubulin-DNA" and "HSP 60-DNA", probably because the two ends of the short-chain probe cannot be anchored with the protein simultaneously due to steric hindrance (FIG. 2B); however, in the case of 100T (Mw about 30KDa) of 100bp sequence, immunoblotting results comparing the former single-head method with the new double-head method show that the double-head method contains the "Aurora A-DNA-Aurora A" complex but does not contain the "tubulin-DNA-tubulin" and the "HSP 60-DNA-HSP 60", but contains the "Aurora A-DNA", "tubulin-DNA" and the "HSP 60-DNA" complex (FIG. 2C), which indicates that the new double-head method can rapidly determine the binding target of the bioactive small molecule by the difference of the proteins at both ends, and when both ends are the same, the double-head method is a specific binding target, and when both ends are different, the double-head method is a non-specific binding target.
4.2 target validation
According to fig. 3, surface plasmon resonance testing showed that the binding response of Tubulin (fig. 3A) and HSP60 (fig. 3B) to MLN8237 increased with increasing concentration of MLN8237, showing a linear relationship, being non-specific binding, as with the other means of target identification.
The above examples are only for illustrating the overall experimental flow by individual methods, but in practice, the method is not limited thereto, and the method for preparing a protein sample is not limited to extraction using Western and IP cell lysates, and includes, but is not limited to, lysis using various lysates (RIPA, NP-40, SDS, 1% Triton X-100, etc.), repeated freeze-thaw, extraction using ultrasound, and the like. Sources of protein samples include, but are not limited to, the use of cultured cells, animal tissues, human specimens, and the like.
The separation of the bioactive small molecule combined target includes, but is not limited to, the above electrophoresis techniques and high performance liquid chromatography, and includes, but is not limited to, paper electrophoresis, polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, agarose gel electrophoresis, isopoint focusing electrophoresis, capillary electrophoresis, ion exchange high performance liquid chromatography, molecular exclusion chromatography, reverse phase high performance liquid chromatography, normal phase high performance liquid chromatography, hydrophilic high performance liquid chromatography, and the like.
Identification of biologically active small molecule binding targets includes, but is not limited to, immunoblotting techniques as described above, including, but not limited to, protein mass spectrometry identification techniques.
Validation of binding of biologically active small molecules to targets includes, but is not limited to, the surface plasmon resonance tests described above, including, but not limited to, isothermal titration calorimetry tests, cellular thermal transition analysis tests, drug affinity target stabilization tests, and the like.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.
Claims (7)
1. A method for identifying a bioactive molecule binding target based on a double-headed photoaffinity probe is characterized by comprising the following steps:
s1 preparation of double-ended photoaffinity probe
Taking groups as units, wherein each group consists of a double-head binding probe and a double-head capture probe; the probe framework of the double-headed binding probe is subjected to base complementary pairing or specific binding with the probe framework of the double-headed capture probe; the two sides of the probe skeleton of the double-head binding probe are combined with the same bioactive molecules, the two sides of the probe skeleton of the double-head capture probe are combined with the same photoaffinity molecules,
two ends of the probe skeleton are subjected to amino modification and then react with the activated ester of the bioactive molecule or the photoaffinity molecule to respectively obtain a double-headed binding probe and a double-headed capture probe, and the double-headed binding probe and the double-headed capture probe are purified and characterized for later use;
s2 protein sample preparation
Extracting a protein sample from a biological sample, and enriching after testing content and purity;
s3, capture, separation and identification of bioactive small molecule binding target
3.1 capture:
respectively hybridizing the double-headed binding probe and the double-headed capture probe, incubating the double-headed photoaffinity probe with a protein sample at a certain concentration, irradiating the double-headed photoaffinity probe and the protein sample under a photoaffinity molecule excitation light source, exciting a light cross-linking reaction to ensure that the capture probe is effectively anchored with a related target protein, and specifically binding the double-headed binding probe and the double-headed capture probe to form a protein-probe-protein covalent complex;
3.2, extraction:
primarily removing unbound free protein from the mixed sample, and drying and dissolving the obtained precipitate; after purifying and enriching the protein-probe-protein complex, removing free probes;
3.3 isolation and identification:
and (3) after the sample in the step (3.2) is dissolved, separating the bioactive small molecule combined target by a certain method, and identifying the target to obtain the bioactive molecule combined target.
2. The method for identifying bioactive molecule-binding targets based on double-ended photoaffinity probes according to claim 1, wherein:
wherein in S1, the probe skeleton is a DNA skeleton, an RNA skeleton or a polypeptide skeleton; the biological molecule comprises a small molecule, an aptamer, a polysaccharide, a polypeptide or a protein; the photoaffinity molecule comprises aromatic azide, diazomethane or benzophenone; and the double-head capture probe is connected with a biological reaction amplification system.
3. The method for identifying bioactive molecule-binding targets based on double-ended photoaffinity probes according to claim 2, wherein:
in S1, the double-headed binding probe is prepared as follows:
firstly, preparing carboxyl or sulfo derivatives of bioactive small molecules, and preparing activated esters of the derivatives by EDC/NHS coupling reaction; secondly, performing amino modification at two ends of the probe skeleton, and further synthesizing a double-end combined probe by utilizing the condensation reaction of amino and activated ester;
the preparation process of the double-head capture probe is as follows:
firstly, preparing carboxyl or sulfo derivatives of photoaffinity molecules, and preparing activated esters of the derivatives by EDC/NHS coupling reaction; secondly, amino modification is carried out at two ends of the probe skeleton, and the double-end capture probe is synthesized by further utilizing the condensation reaction of the amino and activated ester.
4. The method for identifying bioactive molecule-binding targets based on double-ended photoaffinity probes according to claim 2, wherein:
wherein the biological reaction amplification system is a biotinylated base T, and the biotinylated base T is synthesized by a Click reaction.
5. The method for identifying bioactive molecule-binding targets based on double-ended photoaffinity probes according to claim 1, wherein:
wherein, the purification of the double-ended light affinity probe is carried out by adopting the steps of combining ethanol precipitation and chromatographic separation:
ethanol precipitation: measuring the volume of the mixed solution, firstly adding sodium acetate with pH of 5.0 and concentration of 3mol/L in one tenth volume, then adding glycogen with 7.5mg/mL in one tenth volume, then adding absolute ethyl alcohol with 2-3 times volume, uniformly mixing, placing at-80 ℃, and placing for at least 2 hours; taking out a sample, centrifuging 17000g at 4 ℃, discarding the supernatant, adding 70% precooled ethanol into the precipitate, washing by shaking, and centrifuging 17000g at 4 ℃; drying the obtained precipitate, dissolving with triethylamine acetate buffer solution, purifying by preparative chromatography,
and (3) preparing a chromatogram: adopting preparative high performance liquid chromatography; mobile phase: phase A: 0.1mol/L triethylamine acetate buffer solution; phase B: acetonitrile; a chromatographic column: agilent ZORBAX XDB C18 chromatography column; detection wavelength: 210-400 nm; after elution, vacuum freeze-drying is carried out, ultra-pure water is used for redissolving, the concentration of the probe is measured by an ultra-micro fluorescence spectrophotometer, and the purified probe is used for standby after purity and molecular weight determination and characterization.
6. The method for identifying bioactive molecule-binding targets based on double-ended photoaffinity probes according to claim 1, wherein:
wherein, in S2, the protein sample is derived from cultured cells, animal tissues or human samples,
the protein sample extraction method comprises lysate extraction, repeated freeze thawing method or ultrasonic extraction method.
7. The method for identifying bioactive molecule-binding targets based on double-ended photoaffinity probes according to claim 1, wherein:
wherein, in S3, the separation method of the bioactive small molecule combined target comprises electrophoretic separation or high performance liquid chromatography separation;
identification of the bioactive small molecule binding target comprises an immunoblotting technique or a protein mass spectrometry identification technique;
the verification of the combination of the bioactive small molecule and the target comprises a surface plasma resonance test, an isothermal titration calorimetry test, a cell thermal transition analysis test or a drug affinity target stability test.
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