Method for analyzing interaction of strigolactones hormone and receptor protein based on biomass spectrum and Tycho instrument

Abstract

The invention discloses a method for analyzing strigolactones hormone and receptor protein interaction based on a biomass spectrum and a Tycho instrument, and relates to the technical field of biology, the method comprises the following steps: S1, co-incubating a receptor protein solution and a strigolactones compound solution or a blank solvent to respectively obtain a to-be-detected incubation solution and a blank incubation solution; s2, respectively detecting the incubation liquid to be detected and the blank incubation liquid through a Tycho instrument to obtain a thermal denaturation curve of the incubation liquid to be detected and a thermal denaturation curve of the blank incubation liquid; s3, respectively analyzing the incubation liquid to be detected and the blank incubation liquid by using a biomass spectrum to obtain a mass spectrum of the incubation liquid to be detected and a mass spectrum of the blank incubation liquid. And judging whether the strigolactones hormone interacts with the receptor protein or not and whether the hydrolysis reaction occurs or not by comparing the difference between the peak offset of the thermal denaturation curve and the protein signal delta m / z in the mass spectrum. The method not only can be used for interaction detection of plant hormones and receptors, but also can be used for target protein excavation of natural active small molecules.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrumentation. Background Art

[0002] Strigolactones (SLs) are a new type of plant hormone that can regulate plant growth and development and response to adverse environmental stress, and have played a key role in the evolution of plant terrestrial adaptation. Its typical structure contains a tricyclic lactone (ABC ring) and a butenoic acid lactone (D ring), which are connected by an enol ether bond. The atypical strigolactones lack a complete tricyclic lactone, but retain butenoic acid lactone and enol ether bonds, and also have biological activity. The details are as follows: Existing related research has revealed a "substrate-enzyme-active molecule-receptor" hormone recognition mechanism through structural analysis of strigolactones and their receptor complexes. The DWARF14 (D14) receptor protein contained in some plants such as rice, Arabidopsis, tomato, pea, morning glory, and cotton can bind strigolactones in their own ligand binding pockets, and further hydrolyze them into two parts, the ABC ring and the D ring, through the catalytic triad. The ABC ring is released, and the D ring is processed into an active molecule that covalently binds to D14 and induces D14 to change its conformation, thereby interacting with downstream proteins and activating their signal transduction. In this hormone recognition mechanism, changes in protein structure are an important indicator of its function.

[0003] The principle of the Tycho instrument is that during the thermal denaturation process of protein (the thermal denaturation temperature range of protein is 30~90℃), the folding structure of protein changes, and when its endogenous fluorescence (tryptophan / tyrosine) is excited by a wavelength of 280nm, the protein will produce specific emission spectra in different folding states, thereby obtaining an S-shaped curve reflecting the changes in protein folding structure during protein denaturation.

[0004] Existing methods for analyzing the interaction between proteins and small molecules include Biacore technology developed based on optical surface plasmon resonance (SPR), nuclear magnetic resonance technology, isothermal titration calorimetry, cell thermal stability analyzer, microcalorimetry, etc. The above methods have their own advantages when studying unknown compounds or screening unknown targets. In the study of the interaction between trichololactone hormones with relatively clear mechanisms of action and different receptors, the above methods have the disadvantages of high detection cost, long detection time, cumbersome operation, large dosage, and even the need for fluorescent labeling.

[0005] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0006] In response to the above technical problems, the present application provides a method for analyzing the interaction between monocotyledon hormones and receptor proteins based on biomass spectrometry and Tycho instrument. This method can not only be used to detect the interaction between plant hormones and receptors, but also to mine target proteins of natural active small molecules.

[0007] The present application provides a method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument, comprising the following steps: Step S1: co-incubating the receptor protein solution with the strigolactone compound solution to obtain a test incubation solution, and co-incubating the receptor protein solution with a blank solvent to obtain a blank incubation solution; Step S2: respectively testing the incubation solution to be tested and the blank incubation solution by a Tycho instrument to obtain a thermal denaturation curve of the incubation solution to be tested and a thermal denaturation curve of the blank incubation solution, comparing the thermal denaturation curve of the incubation solution to be tested and the thermal denaturation curve of the blank incubation solution, and determining whether the peak value of the thermal denaturation curve of the incubation solution to be tested is offset relative to the peak value of the thermal denaturation curve of the blank incubation solution, and if so, determining that the strigolactone compound interacts with the receptor protein; Step S3: Analyze the incubation solution to be tested and the blank incubation solution respectively by using a biomass spectrometer to obtain a mass spectrum of the incubation solution to be tested and a mass spectrum of the blank incubation solution, and compare the mass spectrum of the blank incubation solution with the mass spectrum of the incubation solution to be tested. If the mass spectrum of the incubation solution to be tested shows the first marker peak relative to the mass spectrum of the blank incubation solution, the strigolactone compound interacts with the receptor protein but no hydrolysis reaction occurs; If the mass spectrum of the incubation solution to be tested shows a second marker peak relative to the mass spectrum of the blank incubation solution, the strigolactone compound interacts with the receptor protein and undergoes a hydrolysis reaction; If the mass spectrum of the incubation solution to be tested is relatively Figure 1 If the reaction is not induced, then the strigolactone compounds do not react with the receptor protein; The first peak is: Δ m / z The value is equal to the molecular weight of the strigolactone compound, or the Δ m / z The values ​​are multiples of the molecular weight of the strigolactone compounds; The second peak is: Δ m / z The value is 96.

[0008] The blank solvent used was a solvent system consisting of 100 mM HEPES (pH 7.4) and 150 mM NaCl.

[0009] Preferably, in step S2: if the Tm peak value of the thermal denaturation curve of the incubation solution to be tested relative to the thermal denaturation curve of the blank incubation solution does not shift, step S3 is required for further confirmation.

[0010] Preferably, the preparation of the incubation solution to be tested comprises: taking the receptor protein solution and the strigolactone compound and placing them in a solvent system to prepare a mixed solution, wherein the final concentration of the receptor protein solution in the mixed solution is 10 μ M, the final concentration of strigolactone compounds in the mixed solution was 200 μ M, after incubation at room temperature in the dark for 1 hour, centrifuge to obtain the incubation solution to be tested.

[0011] Preferably, the detection temperature of the Tycho instrument is 30-95°C, and the excitation light wavelength is 280 nm.

[0012] Preferably, the Tycho instrument analysis comprises: taking 10 μ Place the incubation solution to be tested or the blank incubation solution into the Tycho instrument, set the temperature to rise uniformly from 30°C to 95°C, and the detection time is 3 min.

[0013] Preferably, the liquid phase analysis method includes: the chromatographic column is a C4 chromatographic column specially used for biomacromolecule analysis, with a particle size of 3.6 μ m; the mobile phase was water (+0.1% formic acid) and acetonitrile (+0.1% formic acid) gradient elution; the column temperature was 60°C; the flow rate was 0.4~0.6 ml / min.

[0014] Preferably, the mass spectrometry analysis conditions are: electrospray ion source (ESI); scanning mode: positive ion mode; scanning range: m / z 100~6000; ion spray voltage: 5500V; spray gas (GS1): 50psi; auxiliary heating gas (GS2): 50psi; curtain gas (CUR): 35psi; ionization temperature (TEM): 500℃; declustering voltage (DP): 140V.

[0015] Preferably, the solvent system is composed of 100 mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid) at pH 7.4 (a commonly used hydrogen ion buffer) and 150 mM NaCl.

[0016] Preferably, the monolactone compound is any one of the monolactone monomers: D-ring, GR24, and dGR24.

[0017] The beneficial effects of this application include: 1) The present application provides a method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument. The method can quickly identify the conformational changes of receptor proteins and accurately detect the complex of strigolactone and receptor, and has low cost, small sample analysis amount and short time.

[0018] 2) The present application provides a method for analyzing the interaction between tricholoma lactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument. This method can realize label-free, low-dosage and rapid screening of whether tricholoma lactone hormones interact with receptor proteins, whether hydrolysis reactions occur, and whether the spatial conformation of the protein changes, and no auxiliary means are required to verify the test results again. This method can determine whether the two interact within a minimum of 3 minutes, whether the spatial conformation of the receptor protein changes, and accurately detect the small molecule information bound to the receptor protein within 10 minutes, which is helpful in helping to analyze the interaction mechanism. The method disclosed in the present invention can not only be used for the interaction between plant hormones and receptors, but also can be used for target protein mining of natural active small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The chemical structures and names of the monomers of the strigolactone hormones used in Example 1 of the present application; D-ring, GR24, and dGR24 in the figure are the names of the monomers of the strigolactone hormones with corresponding structural formulas; Figure 2 This is a thermal denaturation curve of the interaction between the strigolactone hormone and the receptor protein D14 in Example 1 of the present application, wherein a is a blank control; b is an incubation solution containing the strigolactone compound D-ring; c is an incubation solution containing the strigolactone compound GR24; d is an incubation solution containing the strigolactone compound dGR24; Figure 3 The mass spectrum of the complex of strigolactone hormone and receptor protein D14 in Example 1 of the present application (the protein signal peak is at RT≈8.1min), wherein a is the total ion current of the liquid-mass analysis of the blank control and the incubation solution to be tested containing strigolactone compounds D-ring, GR24, and dGR24; b is the mass spectrum of the blank control; c is the mass spectrum of the receptor protein D14 and the strigolactone compound D-ring; d is the mass spectrum of the receptor protein D14 and the strigolactone compound GR24; e is the mass spectrum of the receptor protein D14 and the strigolactone compound dGR24; DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention. Example

[0022] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0023] Example 1 Analysis of the interaction between DWARF14 (D14) receptor protein and three strigolactone hormones in plants Preparation of samples to be tested: Take 18 μL of protein solution and 2 μL of strigolactone compounds (the names and structural formulas of the monomers used in subsequent operations are as follows: Figure 1 The mixture was incubated at room temperature in the dark for 1 hour and then centrifuged at 14000 g for 10 min to obtain an incubation solution.

[0024] Tycho instrument analysis: Take 10 μl of the incubation solution and place it in a capillary tube, put it into the Tycho instrument, set the temperature to rise uniformly from 30°C to 95°C, the detection time is 3 minutes, and the excitation light wavelength is 280 nm.

[0025] Biomass spectrometry analysis: 10 μl of incubation solution was placed in a 300 μl liquid phase vial, and a BioZen C4 chromatographic column (150×4.6 mm, 3.6 μm) was used. The flow rate was 0.6 ml / min, the column temperature was 60°C, the mobile phase was pure water (+0.1% formic acid) and acetonitrile (+0.1% formic acid), and the gradient elution conditions were 0~2 min: 15% acetonitrile / water; 2~7 min: 15~60% acetonitrile / water; 7~7.5 min: 60%-90% acetonitrile / water; 7.5~10 min: 90% acetonitrile / water.

[0026] Mass spectrometry analysis conditions are: electrospray ion source (ESI); scanning mode: positive ion mode; scanning range: m / z 100~6000; ion spray voltage: 5500V; spray gas (GS1): 50psi; auxiliary heating gas (GS2): 50psi; curtain gas (CUR): 35psi; ionization temperature (TEM): 500℃; declustering voltage (DP): 140V.

[0027] 1. Thermal denaturation curve and its result analysis: The thermal denaturation curve of the interaction between strigolactone hormones and receptor protein D14 is shown in Figure 2 As shown in a~d, Figure 2 In the middle, a is a blank control, and the treatment method is as follows: the receptor protein D14 and the blank solvent are used to prepare the incubation solution according to the above steps; Figure 2 b is the receptor protein D14 and the structural formula is Figure 1 The thermal denaturation curve of the incubation solution of the strigolactone compound D-ring shown in the figure is obtained by preparing the incubation solution according to the aforementioned steps; Figure 2 c in the figure is the receptor protein D14 and the structural formula is Figure 1 The thermal denaturation curve of the incubation solution of the strigolactone compound GR24 shown in the figure is obtained by preparing the incubation solution according to the aforementioned steps; Figure 2 In the figure, d is the receptor protein D14 and the structural formula is Figure 1 The thermal denaturation curve of the incubation solution of the strigolactone compound dGR24 prepared according to the aforementioned steps is shown.

[0028] The obtained thermal denaturation curve is a first-order singular graph, with the horizontal axis representing temperature and the vertical axis representing the ratio of the fluorescence value of the detected emission light at 350 nm to the fluorescence value at 330 nm.

[0029] Result analysis: Depend on Figure 2 As can be seen from a to d, compared with the control protein (AtD14WT), the peak value of the curve in the AtD14+GR24 group shifted significantly, and the Tm decreased from 58.2℃ to 52.9℃, indicating that GR24 interacted with the receptor protein and caused changes in the spatial conformation of the protein; however, the peak value shifts of the curves in the AtD14+D-ring group and the AtD14+dGR24 group were not obvious, indicating that there may be no interaction, or that there may be interaction but the protein conformation change is not obvious, which requires further confirmation by mass spectrometry.

[0030] 2. Liquid quality analysis results and analysis of results: The results of liquid chromatography-mass spectrometry analysis of the complex of strigolactone hormones and receptor protein D14 are as follows: Figure 3 As shown, Figure 3 Figure a is the total ion current of the blank control and the incubation solution to be tested containing the strigolactone compounds D-ring, GR24, and dGR24, obtained by the above-mentioned analysis method; Figure 3 b is the mass spectrum of blank control; Figure 3 c in the figure is the receptor protein D14 and the structural formula is Figure 1 The mass spectrum of the strigolactone compound D-ring obtained by liquid chromatography-mass spectrometry of the incubation solution prepared according to the aforementioned steps; Figure 3 In the figure, d is the receptor protein D14 and the structural formula is Figure 1 The mass spectrum of the strigolactone compound GR24 obtained by liquid chromatography-mass spectrometry of the incubation solution prepared according to the aforementioned steps; Figure 3 e in the figure is receptor protein D14 and the structural formula is Figure 1 The mass spectrum of the strigolactone compound dGR24 obtained by liquid chromatography-mass spectrometry of the incubation solution prepared according to the aforementioned steps; Result analysis: By comparison Figure 3 The protein molecular weight of the control group AtD14WT in middle b is 29681Da. Figure 3 Medium b and Figure 3 As can be seen in middle c, the mass spectrum signal of the AtD14+D-ring group is the same as that of the control group, indicating that there is no interaction between the two; Figure 3 Another group of Δ appeared in the mass spectrum of AtD14+GR24 group in the middle d m / z 96 protein signals, indicating that AtD14+GR24 interacted and hydrolyzed; comparison Figure 3 Medium b and Figure 3 As can be seen in Figure e, there is no Δ in the mass spectrum of the AtD14+dGR24 group. m / z 96 signals, but Δ m / z The signal of 284 is consistent with the molecular weight of the dGR24 compound, indicating that the two interacted but no hydrolysis reaction occurred.

[0031] This method can be used to accurately analyze whether each monomer of the strigolactone hormone undergoes hydrolysis and interaction with the receptor protein D14. The structure is accurate and reliable, and the detection efficiency is high.

[0032] This method uses the S-shaped curve obtained by the Tycho instrument, and the detection instrument automatically calculates the TI value corresponding to the position where the inflection point appears in the middle of this curve. By comparing the TI values, it can be judged whether the properties, structure, characteristics, functions, etc. of the protein have changed. This feature can be used to design experiments for preliminary detection of protein-molecular interactions.

[0033] In order to better compare the TI values, the S-curve graph is converted into a first-order derivative graph, and the shift of the curve peak is used to determine whether the protein conformation has changed. However, if the curve peak does not shift, it does not necessarily mean that the protein and the small molecule do not interact. It may be that this interaction does not change the spatial conformation of the protein.

[0034] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument, characterized in that: The following steps are involved: Step S1: co-incubating the receptor protein solution with the strigolactone compound solution to obtain a test incubation solution, and co-incubating the receptor protein solution with a blank solvent to obtain a blank incubation solution; Step S2: Analyze the incubation solution to be tested and the blank incubation solution by a Tycho instrument respectively to obtain a thermal denaturation curve of the incubation solution to be tested and a thermal denaturation curve of the blank incubation solution, compare the thermal denaturation curve of the incubation solution to be tested and the thermal denaturation curve of the blank incubation solution, and determine whether the peak value of the thermal denaturation curve of the incubation solution to be tested is offset relative to the peak value of the thermal denaturation curve of the blank incubation solution, and if so, determine that the strigolactone compound interacts with the receptor protein; Step S3: using a biomass spectrometer to analyze the incubation solution to be tested and the blank incubation solution respectively, obtaining a mass spectrum of the incubation solution to be tested and a mass spectrum of the blank incubation solution, and comparing the mass spectrum of the blank incubation solution with the mass spectrum of the incubation solution to be tested. If the mass spectrum of the incubation solution to be tested shows the first marker peak relative to the mass spectrum of the blank incubation solution, the strigolactone compound interacts with the receptor protein but no hydrolysis reaction occurs; If the mass spectrum of the incubation solution to be tested shows a second marker peak relative to the mass spectrum of the blank incubation solution, the strigolactone compound interacts with the receptor protein and undergoes a hydrolysis reaction; If the mass spectrum of the incubation solution to be tested is consistent with the mass spectrum of the blank incubation solution, the strigolactone compound has not reacted with the receptor protein; The first peak is: Δ m / z The value is equal to the molecular weight of the strigolactone compound, or the Δ m / z The values ​​are multiples of the molecular weight of the strigolactone compounds; The second peak is: Δ m / z The value is 96.

2. The method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument according to claim 1, characterized in that: In step S2: if the Tm peak value of the thermal denaturation curve of the incubation solution to be tested relative to the thermal denaturation curve of the blank incubation solution does not shift, further confirmation in step S3 is required.

3. The method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument according to claim 1, characterized in that: The receptor protein solution and the strigolactone compound were respectively placed in a solvent system to prepare a mixed solution. The final concentration of the receptor protein solution in the mixed solution was 10 μ M, the final concentration of strigolactone compounds in the mixed solution was 200 μ M, after incubation at room temperature in the dark for 1 hour, centrifuge to obtain the incubation solution to be tested.

4. The method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument according to claim 1, characterized in that: The analysis temperature of the Tycho instrument is 30~95℃, and the excitation light wavelength is 280nm.

5. The method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument according to claim 1, characterized in that: Tycho instrument analysis includes: taking 10 μ Place the incubation solution to be tested or the blank incubation solution into the Tycho instrument, set the temperature to rise uniformly from 30°C to 95°C, and the detection time is 3 min.

6. The method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument according to claim 1, characterized in that: The biological mass spectrometry analysis described in step S3 is performed by liquid chromatography-mass spectrometry, and the liquid phase analysis method includes: the chromatographic column is a C4 chromatographic column specially used for biological macromolecule analysis, with a particle size of 3.6 μ m; the mobile phase was water (+0.1% formic acid) and acetonitrile (+0.1% formic acid) gradient elution; the column temperature was 60°C; the flow rate was 0.4~0.6 ml / min.

7. The method for analyzing the interaction between strigolactone hormones and receptor proteins based on biomass spectrometry and Tycho instrument according to claim 3, characterized in that: The solvent system consisted of 100 mM HEPES, pH 7.4, and 150 mM NaCl.

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