CN111308006A - LC-MS-based high-throughput high-sensitivity phytohormone detection method - Google Patents
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Abstract
The invention provides a method for detecting phytohormone with high flux and high sensitivity based on LC-MS, which takes 12 isotopes plant hormone as internal standard substance, and separates the plant hormone by reversed phase chromatography, and performing tandem mass spectrometry (LC-MS/MS) analysis by using an internal standard curve method, wherein the 12 isotope phytohormones comprise D-tZ, D-tZR, D-iPR, D-iP, D-BL, D-ABA, D-SA, D-ACC, D-DHZ, D-GA1, D-GA4 and D-CS, and the mass spectrometry adopts an electrospray ionization positive and negative ion (ESI +, ESI-) simultaneous scanning detection mode and a multi-reaction monitoring (MRM) scanning mode.
Description
Technical Field
The invention relates to the field of biotechnology detection, in particular to a method for detecting phytohormone based on LC-MS with high flux and high sensitivity.
Background
Plant hormone (plant hormone), also known as plant natural hormone or plant endogenous hormone, is an organic compound that is induced by plant cells receiving specific environmental signals and that can regulate (promote, inhibit) plant physiological processes at low concentrations. The growth, development and differentiation of plants are regulated and controlled in aspects of cell division and growth, tissue and organ differentiation, flowering and fructification, maturation and aging, dormancy and germination and isolated tissue culture respectively or in a mutually coordinated manner.
Hormones produced in plants are known to be of six major classes, namely auxins (auxins), gibberellins (CTKs), cytokinins (GAs), abscisic acid (ABA), Ethylene (ETH), and Brassinosteroids (BRs). These substances, although contained in a very small amount in plants, have a very large effect and are essential for the life activities of plants, and have a very important meaning in regulating various growth processes and environmental responses of plants.
Most phytohormones do not contain high-sensitivity detection groups, and the matrix is complex, so that the signal-to-noise ratio is difficult to improve; in addition, the concentration distribution of endogenous plant hormones can be as wide as 5-6 orders of magnitude, for example, the contents of auxin, SA, JA and the like are 1-100 ng/g FW (fresh weight), GAs and the like are less than 0.1ng/g FW, BRs and the like are even less than 1pg/g FW, and the measuring method is required to have high sensitivity and wide dynamic measuring range. At present, most methods based on liquid chromatography-mass spectrometry (LC-MS) can only be used for simultaneously measuring one or more phytohormones, the flux is limited, the sensitivity is not high, the popularization and the application are difficult, and a method for detecting the phytohormones at high flux and high sensitivity based on LC-MS is urgently needed.
Disclosure of Invention
The invention aims to provide a high-throughput and high-sensitivity plant hormone detection method based on LC-MS. Has the advantages of high flux, high sensitivity, good selectivity, short analysis time and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
for convenience of writing and reading, the names of hormones in this patent application use acronyms, and the chinese and english controls and acronyms for all hormones are as follows:
a method for detecting phytohormone with high flux and high sensitivity based on LC-MS comprises the following steps:
step S1: preparing isotope internal standard phytohormone mixed solution, respectively weighing various isotope internal standard phytohormones including D-tZ, D-tZR, D-iPR, D-iP, D-BL, D-ABA, D-SA, D-ACC, D-DHZ, D-GA1, D-GA4 and D-CS, respectively adding methanol solution to dissolve the isotope internal standard phytohormone mixed solution into 12 isotope internal standard phytohormone mother solutions with the same mass concentration, uniformly mixing the isotope internal standard phytohormone mother solutions with the same volume, diluting the mixture with 50% methanol water solution to prepare 200ng/mL isotope internal standard phytohormone mixed solution for later use;
step S2: preparing a standard substance phytohormone solution, respectively weighing the standard substance phytohormone, respectively adding a methanol solution to dissolve the standard substance phytohormone solution to prepare a standard substance phytohormone mother solution with the same mass concentration, uniformly mixing various standard substance phytohormone mother solutions with the same volume, and diluting the mixed solution by using a 50% methanol water solution to prepare a 1000ng/mL standard substance phytohormone solution for later use;
step S3: constructing a standard curve, diluting the standard substance phytohormone solution prepared in the step S2 to 1ng/mL in a gradient manner by using a 50% methanol aqueous solution, namely preparing 1000ng/mL, 100ng/mL, 10ng/mL and 1ng/mL standard substance phytohormone solutions with concentration gradients, taking 50uL of each gradient concentration standard substance phytohormone solution, respectively adding 10uL of the isotope internal standard substance phytohormone mixed solution prepared in the step S1, uniformly mixing, respectively adding 940uL of methanol/acetonitrile/water mixed solution, uniformly mixing, standing for 12-24 hours, centrifuging, taking supernatant, placing the supernatant into an Ostro 25mg 96 pore plate, filtering, drying the filtrate by using nitrogen, redissolving by using 200uL of 50% acetonitrile solution, centrifuging, taking the supernatant, and carrying out liquid chromatography-tandem mass spectrometry to obtain the standard curve;
step S4: analyzing a sample, namely adding a plant tissue sample into 10uL of the isotope internal standard product phytohormone mixed solution prepared in the step S1, uniformly mixing, adding 990uL of methanol/acetonitrile/water mixed solution, uniformly mixing, standing for 12-24 hours, centrifuging again, taking supernatant, putting the supernatant into an Ostro 25mg 96 pore plate, filtering, drying filtrate by using nitrogen, redissolving by using 200uL of 50% acetonitrile solution, centrifuging, taking supernatant, performing liquid chromatography-tandem mass spectrometry, and converting an analysis result into a corresponding phytohormone concentration by using a constructed standard curve;
the mass spectrometry conditions in steps S3 and S4 are as follows:
adopting an electrospray ionization positive and negative ion (ESI +, ESI-) simultaneous scanning detection mode and a mass spectrum scanning mode of multi-reaction monitoring (MRM);
positive ion (ESI +) scan mode conditions are as follows: ion source temperature 550 ℃, atomization gas pressure (ion SourceGas 1): 55, assist Gas pressure (ion Source Gas 2): 50, air Curtain gas (CUR)): spray Voltage (ISVF) 30: 4500V;
the negative ion (ESI-) scan mode conditions were as follows: ion source temperature 550 ℃, atomization gas pressure (ion SourceGas 1): 55, assist Gas pressure (ion Source Gas 2): 50, air Curtain gas (CUR)): spray Voltage (ISVF) 30: 4500V.
According to the above scheme, the phytohormone solution as the standard in step S2 includes IAA, tZ, cZ, tZR, cZR, iPR, iP, GA3, JA, BL, ABA, SA, ACC, DHZ, GA1, GA7, GA4, JA-Ile, cis-OPDA, CS, and TY, wherein the objects to be tested cZ and tZ, and cZR and tZR are two groups of isomers, so that the two groups select tZ and tZR as the standard of the respective groups of cZ and tZ, namely the standard curve of the tZ standard for cZ, and the standard curve of the tZR standard for cZR.
According to the scheme, the volume ratio of the methanol/acetonitrile/water mixed liquor in the steps S3 and S4 is 80:19: 1.
According to the above protocol, the centrifugation in steps S3 and S4 was carried out at 14000rcf and 4 ℃ for 15 minutes.
According to the scheme, the step of mixing in S3 and S4 is mixing by vortex.
According to the above scheme, the elution mode of the liquid chromatography in the steps S3 and S4 is mobile phase A: 0.05% aqueous formic acid; mobile phase B: 0.05% formic acid in acetonitrile; the type of the chromatographic column: BEH C181.7 μm,2.1 mm. times.100 mm; the flow rate was 400. mu.L/min, the column temperature was 50 ℃, the sample size was 4. mu.L, and the ratio of mobile phases A and B is given in the following table:
the elution time was 13 min.
According to the above scheme, each ion pair in the mass spectrometry in steps S3 and S4 and the corresponding declustering voltage, collision voltage and collision cell exit voltage parameters are as follows:
the upper part of the upper table is the voltage setting parameter of the electron pair of the standard phytohormone, and the lower part of the upper table is the voltage setting parameter of the electron pair of the internal standard phytohormone.
The invention has the beneficial effects that:
1) the method can finish the pretreatment step of the sample by adopting liquid-liquid extraction, the reversed-phase C18 chromatographic column can effectively separate 21 phytohormones, the pretreatment method is simple, and the experimental operation is simple and convenient;
2) the method can accurately detect important plant hormone substances in plant tissues by quantifying 12 internal standard substances, has high precision and accuracy, can be used for quantitative analysis of the hormones in the plant tissues, has the advantages of high flux, high sensitivity, strong specificity and the like, is simple in experimental operation, provides a reliable detection method for research of the plant hormones, and provides an effective research means for scientific research.
Drawings
FIG. 1 is a TIC graph of a phytohormone standard of the present invention;
Detailed Description
The technical solution of the present invention is described below with reference to the accompanying drawings and examples.
Example 1: measuring the phytohormone of the rice leaves.
The invention provides a high-flux and high-sensitivity detection method for phytohormone based on LC-MS, which comprises the following steps:
step S1: preparing isotope internal standard phytohormone mixed solution, respectively weighing various isotope internal standard phytohormones including D-tZ, D-tZR, D-iPR, D-iP, D-BL, D-ABA, D-SA, D-ACC, D-DHZ, D-GA1, D-GA4 and D-CS, respectively adding methanol solution to dissolve the isotope internal standard phytohormone mixed solution into 12 isotope internal standard phytohormone mother solutions with the concentration of 20ug/mL, respectively taking 10uL of each isotope internal standard phytohormone mother solution to mix uniformly, diluting the mixture with 50% methanol water solution, and preparing 200ng/mL isotope internal standard phytohormone mixed solution for later use.
Step S2: preparing a standard substance phytohormone solution, respectively weighing standard substance phytohormones, wherein the standard substance phytohormones comprise IAA, tZ, cZ, tZR, cZR, iPR, iP, GA3, JA, BL, ABA, SA, ACC, DHZ, GA1, GA7, GA4, JA-Ile, cis-OPDA, CS and TY, respectively adding a methanol solution to dissolve the solutions to prepare a standard substance phytohormone mother solution with the concentration of 20ug/mL, respectively taking 10uL of each standard substance phytohormone mother solution to mix uniformly, diluting the mixed solution with a 50% methanol water solution to prepare a standard substance phytohormone solution with the concentration of 1000ng/mL for later use.
Step S3: constructing a standard curve, diluting the standard substance phytohormone solution prepared in the step S2 to 1ng/mL in a gradient manner by using a 50% methanol aqueous solution, namely preparing the standard substance phytohormone solution with the concentration gradient of 1000ng/mL, 100ng/mL, 10ng/mL and 1ng/mL, taking 50uL of the standard substance phytohormone solution with each gradient concentration, respectively adding 10uL of the isotope internal standard substance phytohormone mixed solution prepared in the step S1, uniformly mixing in a vortex manner, respectively adding a mixed solution of 940uL of methanol/acetonitrile/water in a volume ratio of 80:19:1, uniformly mixing in a vortex manner for 30S, standing for 12-24 hours, centrifuging for 15min at 14000rcf and 4 ℃, taking the supernatant to be placed in an Ostro 25mg 96 pore plate for filtering, drying the filtrate by using nitrogen, redissolving by using a 200uL of 50% acetonitrile solution, centrifuging for 15min at 14000rcf and 4 ℃, taking the supernatant to perform liquid chromatography-tandem mass spectrometry to obtain a standard curve;
step S4: and (2) analyzing a sample, namely adding 80mg of rice leaf powder into 10uL of the isotope internal standard phytohormone mixed solution prepared in the step S1, mixing uniformly by vortex, adding 990uL of mixed solution with methanol/acetonitrile/water of 80:19:1 in volume ratio, mixing uniformly by vortex for 30S, standing for 12-24 hours, centrifuging for 15min at 14000rcf and 4 ℃, putting the supernatant into an Ostro 25mg 96 pore plate for filtering, drying the filtrate by using nitrogen, redissolving by using 200uL of 50% acetonitrile solution, centrifuging for 15min at 14000rcf and 4 ℃, taking the supernatant for liquid chromatography-tandem mass spectrometry, and converting the analysis result into the corresponding phytohormone concentration by using the standard curve constructed in the step S3.
The elution pattern of the liquid chromatography in steps S3 and S4 was mobile phase a: 0.05% aqueous formic acid; mobile phase B: 0.05% formic acid in acetonitrile; the type of the chromatographic column: BEH C181.7 μm,2.1 mm. times.100 mm; the flow rate was 400. mu.L/min, the column temperature was 50 ℃, the sample size was 4. mu.L, and the ratio of mobile phases A and B is given in the following table:
| time (min) | ％A | ％ |
| 0 | 98 | 2 |
| 0.5 | 98 | 2 |
| 1 | 90 | 10 |
| 5 | 80 | 20 |
| 10 | 30 | 70 |
| 10.2 | 5 | 95 |
| 11 | 5 | 95 |
| 11.1 | 98 | 2 |
| 13 | 98 | 2 |
The elution time was 13 min.
The mass spectrometry conditions in steps S3 and S4 are as follows:
adopting an electrospray ionization positive and negative ion (ESI +, ESI-) simultaneous scanning detection mode and a mass spectrum scanning mode of multi-reaction monitoring (MRM);
positive ion (ESI +) scan mode conditions are as follows: ion Source temperature 550 ℃, ion Source Gas 1: 55, ion Source Gas 2: 50, Curtain gas (CUR): 30, ionsaparaty Voltage flowing (ISVF) 4500V;
the negative ion (ESI-) scan mode conditions were as follows: ion Source temperature 550 ℃, ion Source Gas 1: 55, ion Source Gas 2: 50, Curtain gas (CUR): 30, ionSapary Voltage flowing (ISVF) -4500V.
Each ion pair in mass spectrometry and the corresponding declustering voltage, collision voltage and collision cell exit voltage parameters are as follows:
the upper part of the upper table is the voltage setting parameter of the electron pair of the standard phytohormone, and the lower part of the upper table is the voltage setting parameter of the electron pair of the internal standard phytohormone.
The content of each phytohormone in the rice leaf sample is shown in table 1 below.
TABLE 1 content of individual phytohormones in rice leaf samples.
Example two, phytohormone assay of arabidopsis flowers.
The difference between this example and the first example is that the plant tissue sample used in this example is arabidopsis thaliana flower, and the content of each phytohormone in the rice leaf sample is determined and analyzed as shown in table 2 below.
TABLE 2. amount of each phytohormone contained in Arabidopsis thaliana flower samples.
Example three: the stability of the measurement result of the detection method provided by the invention is analyzed.
And establishing a standard curve by adopting an isotope internal standard quantitative method and taking the concentration of the phytohormone standard as an X axis and the peak area ratio of the phytohormone standard to the internal standard as a y axis, and calculating the concentration of the phytohormone in the plant tissue according to the curve. The signal to noise ratio of the peaks according to the characteristic ion MRM chromatogram was greater than 10 as the limit of quantitation (LOQ), and the results are given in table 3 below.
TABLE 3 stability analysis results of phytohormone assay results of one of the examples.
From the results, 21 plant hormones have good linear relation in respective concentration linear ranges, the quantitative requirements are met, and the plant hormones in the plant tissues can be accurately quantified with high flux and high sensitivity.
The above embodiments are only used for illustrating but not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.
Claims (7)
1. A method for detecting plant hormone with high flux and high sensitivity based on LC-MS is characterized by comprising the following steps:
step S1: preparing isotope internal standard phytohormone mixed solution, respectively weighing various isotope internal standard phytohormones including D-tZ, D-tZR, D-iPR, D-iP, D-BL, D-ABA, D-SA, D-ACC, D-DHZ, D-GA1, D-GA4 and D-CS, respectively adding methanol solution to dissolve the isotope internal standard phytohormone mixed solution into 12 isotope internal standard phytohormone mother solutions with the same mass concentration, uniformly mixing the isotope internal standard phytohormone mother solutions with the same volume, diluting the mixture with 50% methanol water solution to prepare 200ng/mL isotope internal standard phytohormone mixed solution for later use;
step S2: preparing a standard substance phytohormone solution, respectively weighing the standard substance phytohormone, respectively adding a methanol solution to dissolve the standard substance phytohormone solution to prepare a standard substance phytohormone mother solution with the same mass concentration, uniformly mixing various standard substance phytohormone mother solutions with the same volume, and diluting the mixed solution by using a 50% methanol water solution to prepare a 1000ng/mL standard substance phytohormone solution for later use;
step S3: constructing a standard curve, diluting the standard substance phytohormone solution prepared in the step S2 to 1ng/mL in a gradient manner by using 50% methanol water solution, taking 50uL of the standard substance phytohormone solution with each gradient concentration, respectively adding 10uL of the isotope internal standard substance phytohormone mixed solution prepared in the step S1, uniformly mixing, respectively adding 940uL of methanol/acetonitrile/water mixed solution, uniformly mixing, standing for 12-24 hours, centrifuging, taking supernatant, placing the supernatant in an Ostro 25mg 96-well plate for filtering, drying the filtrate by using nitrogen, redissolving the filtrate by using 200uL of 50% acetonitrile solution, centrifuging, taking the supernatant, and performing liquid chromatography-tandem mass spectrometry to obtain the standard curve;
step S4: analyzing a sample, namely adding a plant tissue sample into 10uL of the isotope internal standard product phytohormone mixed solution prepared in the step S1, uniformly mixing, adding 990uL of methanol/acetonitrile/water mixed solution, uniformly mixing, standing for 12-24 hours, centrifuging again, taking supernatant, putting the supernatant into an Ostro 25mg 96 pore plate, filtering, drying filtrate by using nitrogen, redissolving by using 200uL of 50% acetonitrile solution, centrifuging, taking supernatant, performing liquid chromatography-tandem mass spectrometry, and converting an analysis result into a corresponding phytohormone concentration by using a constructed standard curve;
the mass spectrometry conditions in steps S3 and S4 are as follows:
adopting an electrospray ionization positive and negative ion simultaneous scanning detection mode and a mass spectrum scanning mode of multi-reaction monitoring;
the positive ion scan mode conditions were as follows: ion source temperature: 550 ℃, atomization pressure: 55, auxiliary air pressure: 50, air curtain gas: 30, spray voltage: 4500V;
the negative ion scan mode conditions were as follows: ion source temperature 550 ℃, atomization pressure: 55, auxiliary air pressure: 50, air curtain pressure: 30, spray voltage: 4500V.
2. The LC-MS-based high-throughput high-sensitivity plant hormone detection method according to claim 1, wherein the standard plant hormone solution in the step S2 comprises IAA, tZ, cZ, tZR, cZR, iPR, iP, GA3, JA, BL, ABA, SA, ACC, DHZ, GA1, GA7, GA4, JA-Ile, cis-OPDA, CS and TY.
3. The LC-MS-based high-throughput high-sensitivity plant hormone detection method of claim 1, wherein the volume ratio of the methanol/acetonitrile/water mixture in the steps S3 and S4 is 80:19: 1.
4. The LC-MS-based method for high-throughput and high-sensitivity detection of plant hormones as claimed in claim 1, wherein the centrifugation in steps S3 and S4 is performed at 14000rcf and 4 ℃ for 15 minutes.
5. The LC-MS-based high-throughput high-sensitivity plant hormone detection method of claim 1, wherein the step S3 and S4 are performed by vortex mixing.
6. The LC-MS-based method for high-throughput and high-sensitivity detection of plant hormones as claimed in claim 1, wherein the elution manner of the liquid chromatography in steps S3 and S4 is mobile phase A: 0.05% aqueous formic acid; mobile phase B: 0.05% formic acid in acetonitrile; the type of the chromatographic column: BEH C181.7 μm,2.1 mm. times.100 mm; the flow rate was 400. mu.L/min, the column temperature was 50 ℃, the sample size was 4. mu.L, and the ratio of mobile phases A and B is given in the following table:
The elution time was 13 min.
7. The LC-MS-based high-throughput high-sensitivity plant hormone detection method according to claim 1, wherein the ion pairs and their corresponding declustering voltage, collision voltage and collision cell exit voltage parameters in the mass spectrometry of the steps S3 and S4 are as follows:
the upper part of the upper table is the voltage setting parameter of the electron pair of the standard phytohormone, and the lower part of the upper table is the voltage setting parameter of the electron pair of the internal standard phytohormone.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116297964A (en) * | 2023-03-23 | 2023-06-23 | 浙江石原金牛生物科技有限公司 | LC-MS detection method for various plant hormones in liquid seaweed fertilizer |
| CN116297964B (en) * | 2023-03-23 | 2023-09-01 | 浙江石原金牛生物科技有限公司 | LC-MS detection method for various plant hormones in liquid seaweed fertilizer |
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