A method for analyzing the interaction between strigolactone hormones and receptor proteins based on mass spectrometry and Tycho instrument

Abstract

The present application discloses a method for analyzing the interaction between strigolactone hormones and receptor proteins based on mass spectrometry and Tycho instrument, which relates to the field of biotechnology and includes the following steps: Step S1: Incubate the receptor protein solution with the strigolactone compound solution or the blank solvent to obtain the test incubation solution and the blank incubation solution respectively; Step S2: Detect the test incubation solution and the blank incubation solution respectively by the Tycho instrument to obtain the thermal denaturation curve of the test incubation solution and the thermal denaturation curve of the blank incubation solution; Step S3: Analyze the test incubation solution and the blank incubation solution respectively using mass spectrometry to obtain the mass spectrometry of the test incubation solution and the mass spectrometry of the blank incubation solution. By comparing the peak shift of the thermal denaturation curve and the difference in protein signals in the mass spectrometry Δ m / z to determine whether the strigolactone hormone interacts with the receptor protein and whether a hydrolysis reaction occurs. This method can not only be used for the detection of the interaction between plant hormones and receptors, but also for the discovery of target proteins of natural active small molecules.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and particularly to a method for analyzing the interaction between strigolactone hormones and receptor proteins based on mass spectrometry and Tycho instrument. Background Art

[0002] Strigolactones (SLs) are a new type of plant hormone that can regulate plant growth and development and stress responses, and play a key role in the terrestrial adaptive evolution of plants. Its typical structure contains a tricyclic lactone (ABC ring) and a butenolide (D ring), which are connected by an enol ether bond. Atypical strigolactones lack a complete tricyclic lactone, but retain the butenolide and enol ether bond and also have biological activity. Specifically, it is shown as follows:

[0003]

[0004] Existing related research has revealed a "substrate-enzyme-active molecule-receptor" hormone recognition mechanism through the structural analysis of strigolactones and their receptor complexes. The DWARF14 (D14) receptor proteins contained in some plants such as rice, Arabidopsis thaliana, 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 a catalytic triad. The ABC ring is released, while the D ring is processed into an active molecule and covalently binds to D14, inducing D14 to change its own conformation, thereby interacting with downstream proteins and activating its signal transduction. In this hormone recognition mechanism, the change in protein structure is an important indicator of its function.

[0005] The principle of the Tycho instrument is that during the thermal denaturation process of proteins (the protein thermal denaturation temperature range is 30 - 90 °C), the folding structure of proteins changes. When its endogenous fluorescence (tryptophan / tyrosine) is excited by a wavelength of 280 nm, proteins will have specific emission spectra in different folding states, thereby obtaining an S-shaped curve that reflects the change in the protein folding structure during the protein denaturation process.

[0006] Existing methods for analyzing the interaction between proteins and small molecules include the Biacore technology developed based on optical surface plasmon resonance (SPR), nuclear magnetic resonance technology, isothermal titration calorimetry, cell thermal stability analyzer, microcalorimetric surging technology, and so on. The above methods each have their own advantages when studying unknown compounds or screening unknown targets. In the study of the interaction relationship between strigolactone hormones with clear action mechanisms and different receptors, the above methods have deficiencies such as high detection cost, long detection time, cumbersome operation, large consumption, and even the need for fluorescence labeling.

[0007] The information disclosed in the background art section is only intended to enhance the overall understanding of the background of the present invention and should not be construed as an admission or any form of implication that such information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0008] The present application provides a method for analyzing the interaction between strigolactone hormones and receptor proteins based on mass spectrometry and a Tycho instrument. This method can not only be used for detecting the interaction between plant hormones and receptors, but also for mining target proteins of natural active small molecules.

[0009] The present application provides a method for analyzing the interaction between strigolactone hormones and receptor proteins based on mass spectrometry and a Tycho instrument, comprising the following steps:

[0010] Step S1: Co-incubate the receptor protein solution with the strigolactone compound solution to obtain a test incubation solution, and co-incubate the receptor protein solution with a blank solvent to obtain a blank incubation solution;

[0011] Step S2: Detect the test incubation solution and the blank incubation solution respectively by a Tycho instrument to obtain a thermal denaturation curve of the test incubation solution and a thermal denaturation curve of the blank incubation solution. Compare the thermal denaturation curve of the test incubation solution and the thermal denaturation curve of the blank incubation solution, and determine whether the peak value of the thermal denaturation curve of the test incubation solution is shifted relative to the peak value of the thermal denaturation curve of the blank incubation solution. If so, it is determined that the strigolactone compound interacts with the receptor protein;

[0012] Step S3: Analyze the test incubation solution and the blank incubation solution respectively using mass spectrometry to obtain a mass spectrum of the test incubation solution and a mass spectrum of the blank incubation solution. Compare the mass spectrum of the blank incubation solution and the mass spectrum of the test incubation solution

[0013] If a first marker peak appears in the mass spectrum of the test incubation solution relative to the mass spectrum of the blank incubation solution, then the strigolactone compound interacts with the receptor protein but does not undergo hydrolysis reaction;

[0014] If a second marker peak appears in the mass spectrum of the test incubation solution relative to the mass spectrum of the blank incubation solution, then the strigolactone compound interacts with the receptor protein and undergoes hydrolysis reaction;

[0015] If the mass spectrum of the test incubation solution is relative to the mass spectrum of the blank incubation solution Figure 1 is the same, then the strigolactone compound does not react with the receptor protein;

[0016] The first marker peak is: Δ in the mass spectrum corresponding to the test incubation solution m / z The numerical value is equal to the molecular weight of the strigolactone compound, or Δ in the mass spectrum corresponding to the test incubation solution m / zThe value is a multiple of the molecular weight of the strigolactone compound;

[0017] The second marker peak is: Δ in the mass spectrum corresponding to the test incubation solution m / z The value is 96.

[0018] The blank solvent used is a solvent system: 100 mM HEPES (pH 7.4) and 150 mM NaCl.

[0019] Preferably, in step S2: If the Tm peak of the thermal denaturation curve of the test incubation solution does not shift relative to the thermal denaturation curve of the blank incubation solution, step S3 is required for further confirmation.

[0020] Preferably, the preparation of the test incubation solution includes: separately taking the receptor protein solution and the strigolactone compound and placing them in a solvent system to prepare a mixed solution. The final concentration of the receptor protein solution in the mixed solution is 10 μ M, and the final concentration of the strigolactone compound in the mixed solution is 200 μ M. After incubating in the dark at room temperature for 1 hour, centrifuge to obtain the test incubation solution.

[0021] Preferably, the detection temperature of the Tycho instrument is 30 - 95 °C, and the excitation light wavelength is 280 nm.

[0022] Preferably, the analysis by the Tycho instrument includes: taking 10 μ μL of the test incubation solution or the blank incubation solution, putting it into the Tycho instrument, setting the temperature to rise uniformly from 30 °C to 95 °C, and the detection time is 3 min.

[0023] Preferably, the liquid phase analysis method includes: the chromatographic column is a dedicated C4 chromatographic column for biomacromolecule analysis, with a particle size of 3.6 μ μm; the mobile phase is gradient elution of water (+0.1% formic acid) and acetonitrile (+0.1% formic acid); the column temperature is 60 °C; the flow rate is 0.4 - 0.6 ml / min.

[0024] Preferably, the mass spectrometry analysis conditions are: electrospray ionization source (ESI); scan mode: positive ion mode; scan range: m / z 100 - 6000; ion spray voltage: 5500 V; nebulizing gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi; curtain gas (CUR): 35 psi; ionization temperature (TEM): 500 °C; declustering voltage (DP): 140 V.

[0025] Preferably, the solvent system is composed of 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, a commonly used hydrogen ion buffer) at pH 7.4 and 150 mM NaCl.

[0026] Preferably, the strigolactone compound is any one of strigolactone monomers: D-ring, GR24, and dGR24.

[0027] The beneficial effects that can be produced by this application include:

[0028] 1) A method for analyzing the interaction between strigolactone hormones and receptor proteins based on mass spectrometry and Tycho instrument provided by this application. This method can quickly identify the conformational changes of receptor proteins, accurately detect the complex of strigolactone and receptors, and has low cost, small sample analysis consumption, and short time consumption.

[0029] 2) A method for analyzing the interaction between strigolactone hormones and receptor proteins based on mass spectrometry and Tycho instrument provided by this application. This method can achieve label-free, low-consumption, and rapid screening of whether strigolactone hormones and receptor proteins interact, whether hydrolysis reactions occur, and whether the protein spatial conformation changes, and there is no need for auxiliary means to verify the detection results again. This method can judge whether the interaction occurs between the two and whether the spatial conformation of the receptor protein changes within at least 3 minutes, and accurately detect the small molecule information bound to the receptor protein within 10 minutes, which is beneficial to helping 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 for the discovery of target proteins of natural bioactive small molecules. Description of the Drawings

[0030] Figure 1 It is the chemical structures and names of each monomer of the strigolactone hormones used in Example 1 of this application; in the figure, D-ring, GR24, and dGR24 are all the names of strigolactone hormone monomers with corresponding structural formulas;

[0031] Figure 2 It is the thermal denaturation curve of the interaction between strigolactone hormones and receptor protein D14 in Example 1 of this application, where a is the blank control; b is the incubation solution containing strigolactone compound D-ring; c is the incubation solution containing strigolactone compound GR24; d is the incubation solution containing strigolactone compound dGR24;

[0032] Figure 3Mass spectrometry diagram of the strigolactone hormone and receptor protein D14 complex in Example 1 of this application (the protein signal peak is at RT≈8.1 min). Among them, a is the total ion chromatogram of the liquid mass spectrometry analysis of the blank control and the test incubation solution containing the strigolactone compound D-ring, GR24, and dGR24; b is the mass spectrometry diagram of the blank control; c is the mass spectrometry diagram of receptor protein D14 and the strigolactone compound D-ring; d is the mass spectrometry diagram of receptor protein D14 and the strigolactone compound GR24; e is the mass spectrometry diagram of receptor protein D14 and the strigolactone compound dGR24; Detailed implementation manners

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited in any way. Any transformation or improvement made based on the teachings of the present invention falls within the protection scope of the present invention. Embodiment

[0034] In the following embodiments, the materials and instruments used are obtained from commercial channels unless otherwise specified; the detection methods used are existing methods unless otherwise specified.

[0035] Embodiment 1 Analysis of the interaction between DWARF14 (D14) receptor protein in plants and three strigolactone hormones

[0036] Preparation of the test sample: Take 18 μL of protein solution and 2 μL of strigolactone compound (the names and structural formulas of each monomer used in the subsequent operations are as Figure 1 shown) and place them in a 1.5 ml centrifuge tube. The solvent system preset in the centrifuge tube is a mixed solution of 100 mM HEPES (pH 7.4) and 150 mM NaCl. The final concentrations of the receptor protein and the strigolactone compound in the prepared mixed solution are 10 μM and 200 μM respectively. After incubating in the dark at room temperature for 1 hour, centrifuge at 14000 g for 10 min to obtain the incubation solution.

[0037] Analysis by Tycho instrument: Take 10 μl of the incubation solution and place it in a capillary, then put it into the Tycho instrument. The temperature is set to rise uniformly from 30 °C to 95 °C, the detection time is 3 min, and the excitation light wavelength is 280 nm.

[0038] Biomass spectrometry analysis: Take 10 μl of the incubation solution and place it in a 300 μl liquid phase vial. Use a BioZen C4 chromatographic column (150×4.6 mm, 3.6 μm), with a flow rate of 0.6 ml / min, a column temperature of 60 °C. The mobile phase is pure water (+0.1% formic acid) and acetonitrile (+0.1% formic acid). The gradient elution conditions are as follows: 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.

[0039] The mass spectrometry analysis conditions are: electrospray ionization source (ESI); scanning mode: positive ion mode; scanning range: m / z 100 - 6000; ion spray voltage: 5500 V; nebulizing gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi; curtain gas (CUR): 35 psi; ionization temperature (TEM): 500 °C; declustering potential (DP): 140 V.

[0040] 1. Thermal denaturation curve and its result analysis:

[0041] The thermal denaturation curves of the interaction between strigolactone hormones and receptor protein D14 are as shown in Figure 2 a - d below. Figure 2 In a, it is a blank control, and the treatment method is: the receptor protein D14 and the blank solvent are used to prepare the incubation solution according to the aforementioned steps; Figure 2 In b, it is the thermal denaturation curve obtained from the incubation solution prepared by the receptor protein D14 and the strigolactone compound D - ring with the structure as shown in Figure 1 according to the aforementioned steps; Figure 2 In c, it is the thermal denaturation curve obtained from the incubation solution prepared by the receptor protein D14 and the strigolactone compound GR24 with the structure as shown in Figure 1 according to the aforementioned steps; Figure 2 In d, it is the thermal denaturation curve obtained from the incubation solution prepared by the receptor protein D14 and the strigolactone compound dGR24 with the structure as shown in Figure 1 according to the aforementioned steps.

[0042] The obtained thermal denaturation curve is a first - order single - number graph. The abscissa is temperature, and the ordinate is obtained by dividing the fluorescence value of the detected emitted light at 350 nm by the fluorescence value at 330 nm.

[0043] Result analysis:

[0044] From Figure 2As shown in Figures a - d, compared with the control protein (AtD14WT), the peak of the curve in the AtD14 + GR24 group shifted significantly, and the Tm decreased from 58.2 °C to 52.9 °C, indicating that GR24 interacted with the receptor protein and caused a change in the protein's spatial conformation. However, the peak shift of the curves in the AtD14 + D - ring group and the AtD14 + dGR24 group was not obvious, suggesting that there may be no interaction, or there may be an interaction but the protein conformation change is not obvious, and further confirmation by mass spectrometry is needed.

[0045] 2. LC - MS analysis results and their analysis:

[0046] The LC - MS analysis results of the strigolactone hormone - receptor protein D14 complex are as Figure 3 shown. Figure 3 In Figure a, it is the total ion chromatogram of the LC - MS analysis of the blank control and the test incubation solutions containing the strigolactone compounds D - ring, GR24, and dGR24, obtained by analyzing according to the aforementioned method. Figure 3 In Figure b, it is the mass spectrum of the blank control. Figure 3 In Figure c, it is the mass spectrum obtained by LC - MS analysis of the incubation solution prepared by the receptor protein D14 and the strigolactone compound D - ring with the structure as Figure 1 shown according to the aforementioned steps. Figure 3 In Figure d, it is the mass spectrum obtained by LC - MS analysis of the incubation solution prepared by the receptor protein D14 and the strigolactone compound GR24 with the structure as Figure 1 shown according to the aforementioned steps. Figure 3 In Figure e, it is the mass spectrum obtained by LC - MS analysis of the incubation solution prepared by the receptor protein D14 and the strigolactone compound dGR24 with the structure as Figure 1 shown according to the aforementioned steps.

[0047] Results analysis:

[0048] By comparison, Figure 3 in Figure b, the molecular weight of the control group AtD14WT protein is 29681 Da. Comparing Figures b and Figure 3 c, it can be seen that the mass spectrum signals in the AtD14 + D - ring group are the same as those in the control group, indicating that there is no interaction between the two. Figure 3 In Figure d, another set of Δ Figure 3 96 protein signals appear in the mass spectrum of the AtD14 + GR24 group, indicating that an interaction and hydrolysis reaction occurred in AtD14 + GR24. Comparing Figures b and m / z e, it can be seen that no Δ Figure 3 96 signal appears in the mass spectrum of the AtD14 + dGR24 group, but a Δ Figure 3 m / z m / 96 signal appears, but a Δ m / zThe 284 signal, which is consistent with the molecular weight of the dGR24 compound, indicates that the two interacted, but no hydrolysis reaction occurred.

[0049] Using this method, it is possible to accurately analyze whether each monomer of strigolactone hormones undergoes hydrolysis and interaction reactions with the receptor protein D14, with accurate and reliable structure and high detection efficiency.

[0050] This method uses the S-shaped curve obtained by the Tycho instrument. The detection instrument automatically locates the position in the middle of this curve where an inflection point appears, and calculates the TI value corresponding to this position. By comparing the TI values, it is possible to determine whether the properties, structure, characteristics, functions, etc. of the protein have changed. This characteristic can be used to design experiments for preliminarily detecting intermolecular interactions of proteins.

[0051] To better compare the TI values, the S-shaped curve graph is converted into a first derivative graph, and whether the protein conformation has changed is judged by the shift of the curve peak. However, the fact that the curve peak does not shift does not necessarily mean that the protein does not interact with the small molecule. It may be that this interaction relationship does not change the spatial conformation of the protein.

[0052] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within 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 Tycho instrument respectively to obtain the thermal denaturation curve of the incubation solution to be tested and the 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 significantly offset relative to the peak value of the thermal denaturation curve of the blank incubation solution. If so, it is determined that the strigolactone compound interacts with the receptor protein; the significant offset is T m The change value is greater than 5.3°C; if not, further confirmation is required in step S3; 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; mobile phase A was water + 0.1% formic acid, and mobile phase B was acetonitrile + 0.1% formic acid, for gradient elution; column temperature was 60°C; 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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