CN111351877A - Tissue energy metabolism substance analysis method based on UPLC-MSMS - Google Patents
Tissue energy metabolism substance analysis method based on UPLC-MSMS Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
Abstract
The invention aims to provide a UPLC-MSMS-based method for analyzing tissue energy metabolites, which is characterized in that a liquid chromatography-mass spectrometry combined technical means is adopted to pertinently and quantitatively detect key metabolites participating in an energy metabolism pathway, particularly key metabolites in TCA (ternary content addressable memory) circulation, glycolysis and oxidative phosphorylation pathways, the method is simple, rapid and accurate, the complexity of derivatization experiments is avoided, the separation is carried out by using a liquid chromatography during detection, and meanwhile, a high-specificity and high-sensitivity analysis strategy of a mass spectrometry multi-reaction monitoring mode is adopted, so that the content of up to 20 important energy metabolites can be obtained through one-time sample injection analysis, and the UPLC-MSMS-based method has the advantages of high precision, simple pretreatment, high sensitivity, good repeatability and the like.
Description
Technical Field
The invention relates to the field of biotechnology detection, in particular to a tissue energy metabolite analysis method based on UPLC-MSMS.
Background
Energy metabolism maintains the most essential vital activities of a living body, such as plant stress and animal diseases, often accompanied by severe metabolic disorders, and most of them attributed to energy metabolism disorders. Energy metabolism also affects immune cell function and fate, and is associated with disease resistance and immune responses. Meanwhile, the modification group for protein phosphorylation comes from ATP, so that energy metabolism abnormality can further cause the change of an upstream protein phosphorylation pathway, and various physiological and pathological functions of organisms are seriously influenced. Energy metabolism processes in the organism mainly include the tricarboxylic acid cycle, glycolytic pathway, and oxidative phosphorylation processes. The tricarboxylic acid cycle (also known as the citric acid cycle or the TCA cycle or the Krebs cycle) is a ubiquitous metabolic pathway in aerobic organisms, distributed in mitochondria; is the final metabolic pathway of three major nutrients (carbohydrates, lipids, amino acids); but also as the hub of carbohydrate, lipid and amino acid metabolic link. The glycolytic pathway (also called EMP pathway) is a series of reactions that degrade glucose and glycogen to pyruvate with the production of ATP, a pathway of glucose degradation that is ubiquitous in all biological organisms. The EMP pathway provides a certain amount of energy to the organism, and the intermediate products provide raw materials for biosynthesis. The oxidative phosphorylation process, which is present in mitochondria or bacteria of eukaryotic cells, is a coupling reaction in which energy released when a substance is oxidized in vivo is supplied to ADP and inorganic phosphorus to synthesize ATP. Therefore, the content of the metabolites related to the energy metabolism pathways of various biological sample species can be comprehensively and intuitively analyzed, and the method has positive guiding significance for researching the physiology and the pathology of diseases related to the energy metabolism disorder.
At present, three main analysis strategies are provided for key substances of an energy metabolism pathway, one is that a sample to be detected is subjected to derivatization treatment and then is subjected to gas chromatography-triple quadrupole mass spectrometry combined analysis (Chinese patent CN 106855551A), and because the method adopts gas chromatography for separation, a methoxylamine pyridine solution is firstly used for resuspending the sample before detection, and then N-methyl-N- (trimethylsilyl) trifluoroacetamide is added for derivatization reaction to carry out chromatographic separation, so that the preparation time of the sample is too long, and only the derivatization reaction experiment takes at least 3 hours; the second method is to use a high-pressure liquid chromatograph with a diode array detector for detection (Chinese patent CN 108303475A), the diode array detector adopted by the method has poor sensitivity and can only reach the level of mu g/mL, the method qualitatively and accurately determines the substance to be detected by comparing the chromatographic retention time with a standard substance, and if the detected sample is too complex, an interfering substance can be generated to influence the qualitative analysis of the substance, so the method cannot be an energy substance detection method which can be widely applied to various sample types; the third method is to adopt a commercial kit to detect related metabolites, the method has high detection cost, complicated experimental steps and extremely high requirement on the operation proficiency of experimenters, and each experiment can only detect a single substance, so that the method has strong limitation.
Disclosure of Invention
Based on the problems in the prior art, the invention aims to provide a UPLC-MSMS-based method for analyzing tissue energy metabolites, which adopts a liquid chromatography-mass spectrometry combined technical means to specifically and quantitatively detect key metabolites participating in an energy metabolism pathway, particularly key metabolites in TCA (ternary content addressable memory) circulation, glycolysis and oxidative phosphorylation pathways, is simple, rapid and accurate, avoids the complexity of derivatization experiments, is separated by using a liquid chromatography during detection, adopts a high-specificity and high-sensitivity analysis strategy of a mass spectrometry multi-reaction monitoring mode, can obtain the content of up to 20 important energy metabolites through one-time sample injection analysis, and has the advantages of high precision, simple pretreatment, high sensitivity, good repeatability and the like.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the tissue energy metabolism substance analysis method based on the UPLC-MSMS comprises the following steps:
s1, constructing a standard curve, respectively weighing energy metabolite standard substances, preparing an energy metabolite standard substance mixed solution with a series of gradient concentrations by using 50% acetonitrile aqueous solution, and directly performing the steps S3 and S4 on the standard substance mixed solution to obtain the standard curve;
step S2, preparing a sample, namely taking a tissue sample to be detected, adding water to prepare a tissue homogenate, adding a methanol/acetonitrile mixed solution, carrying out vortex mixing, carrying out ultrasonic treatment in ice bath, incubating precipitated protein, carrying out high-speed centrifugation to obtain a supernatant, and carrying out vacuum drying for later use;
and step S3, carrying out liquid chromatographic analysis, namely, redissolving the dried sample obtained in the step S2 by using an acetonitrile aqueous solution, centrifuging and taking the supernatant to carry out liquid chromatographic analysis, wherein the chromatographic conditions are as follows:
injecting sample at 4 deg.C, column temperature 45 deg.C, and flow rate 300 μ L/min; the liquid phase gradient was as follows: the liquid B is linearly changed from 95% to 76% in 0-12 min; the liquid B is linearly changed from 76% to 47% in 12-12.1 min; the liquid B is linearly changed from 47 percent to 95 percent in 12.1-15.1min and is maintained at 95 percent in 15.1-22 min;
step S4, performing mass spectrometry, wherein the sample subjected to chromatographic separation in the step S3 directly enters the mass spectrometry under the conditions that an ESI source is adopted and the ion source temperature (source temperature) is 450 ℃; atomization Gas pressure (ion Source Gas1(Gas 1)): 45, a first step of; assist Gas pressure (Ion Source Gas2(Gas 2)): 45, a first step of; air Curtain gas (Curtain): 30, of a nitrogen-containing gas; spray Voltage (ionSapary Voltage flowing (ISVF)): 4500V, using multiple reaction monitoring mode detection.
According to the above scheme, the concentration range of the series of gradients in the step S1 is 1-50000ng/mL, and the energy metabolite standard includes aconitic acid, adenosine diphosphate, α -ketoglutaric acid, adenosine monophosphate, adenosine triphosphate, citric acid, cyclic adenosine monophosphate, flavin mononucleotide, fumaric acid, guanosine 5' -diphosphate, guanosine 5' -monophosphate, guanosine 5' -triphosphate, isocitric acid, malic acid, nicotinamide adenine dinucleotide phosphate, phosphoenolpyruvic acid, pyruvic acid, succinic acid, and triphenyl phosphate.
According to the scheme, the water and methanol/acetonitrile mixed solution in the step S2 is pre-cooled to a temperature of 4 ℃, the water is ultrapure water, and the methanol/acetonitrile mixed solution is prepared by mixing the following components in a volume ratio of 1: 1, the ultrasonic treatment is 350W treatment for 20min, and the incubation of the precipitated protein is carried out for 1h at the temperature of minus 20 ℃.
According to the above protocol, the centrifugation of step S2 and step S3 is 14000rcf 4 ℃ for 15 min.
According to the above scheme, the dried sample is redissolved with 50% acetonitrile in the step S3; the solution A in the step S3 is 10mM ammonium acetate water solution, pH 9.2; the solution B is 10mM ammonium acetate water solution dissolved in 85% acetonitrile water solution and has a pH value of 9.2.
According to the above scheme, the metabolite quantitative ion pair, the qualitative ion pair and the corresponding collision energy information of the mass spectrum analysis in step S4 are as follows:
an ion pair with good specificity is selected as a quantitative ion pair and is supplemented with a qualitative ion pair, and DP in the table is Declustering potential (Declustering potential), CE is Collision energy (Collision energy), and CXP is Collision cell exit potential (Collision cell exit potential).
The invention has the beneficial effects that:
1) the method is simple, rapid and accurate by adopting a liquid chromatography-mass spectrometry combined technical means, and the complexity of a derivatization experiment is avoided;
2) by adopting a high-specificity and high-sensitivity analysis strategy of a mass spectrum multi-reaction monitoring mode, the content of up to 20 important energy metabolites can be obtained through one-time sample injection analysis, and the method has the advantages of high precision, simple pretreatment, high sensitivity, good repeatability and the like.
Drawings
FIG. 1 is an energy metabolism standard mixture XIC profile in the examples;
FIG. 2 is a TIC map of a mouse heart tissue sample of the examples.
The attached drawings are experimental result graphs which inevitably appear in the embodiment, the result graph of each experiment is changed due to the difference of each actual experiment, and the repeated implementation of the technical scheme cannot be influenced due to unclear characters in the graphs.
Detailed Description
The technical solution of the present invention is described below with reference to the accompanying drawings and examples.
The tissue energy metabolism substance analysis method based on UPLC-MSMS, in the embodiment, the sample to be detected is mouse heart, includes the following steps:
step S1, constructing a standard curve, respectively weighing 20 energy metabolite standards, preparing an energy metabolite standard mixed mother liquor by using 50% acetonitrile aqueous solution, wherein the concentration of each energy metabolite standard in the mixed mother liquor is 50 mu M, and then diluting the energy metabolite standard mixed mother liquor into energy metabolite standard mixed liquor with the following series of gradient concentrations by using 50% acetonitrile aqueous solution, wherein the energy metabolite standard mixed liquor is 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL, 2500ng/mL, 5000ng/mL, 10000ng/mL, 25000ng/mL and 50000 ng/mL;
the energy metabolite standard includes aconitic acid, adenosine diphosphate, α -ketoglutaric acid, adenosine monophosphate, adenosine triphosphate, citric acid, cyclic adenosine monophosphate, flavin mononucleotide, fumaric acid, guanosine 5' -diphosphate, guanosine 5' -guanosine, guanosine 5' -triphosphate, isocitric acid, malic acid, nicotinamide adenine dinucleotide phosphate, phosphoenolpyruvic acid, pyruvic acid, succinic acid, and triphenyl phosphate;
the obtained standard mixture is directly processed to step S3 to obtain a standard curve, and the XIC spectrum of the standard mixture is shown in fig. 1.
Step S2, preparing a sample, namely weighing 40mg of mouse heart samples, adding 200 mu L of ultrapure water at 4 ℃ to prepare tissue homogenate, and adding 800 mu L of methanol/acetonitrile mixed solution at 4 ℃, wherein the volume ratio of the methanol/acetonitrile mixed solution is 1: 1, vortex mixing, treating with 350W ultrasonic wave in ice bath for 20min, incubating at-20 deg.C for 1h to precipitate protein, centrifuging at 14000rcf and 4 deg.C for 15min, collecting supernatant, and vacuum drying.
Step S3 liquid chromatography, 100 μ L of 50% acetonitrile in water is used to reconstitute the dried sample obtained in step S2, the supernatant is centrifuged and subjected to liquid chromatography, the chromatographic conditions are as follows:
injecting sample at 4 deg.C, column temperature 45 deg.C, and flow rate 300 μ L/min; the liquid phase gradient was as follows: the liquid B is linearly changed from 95% to 76% in 0-12 min; the liquid B is linearly changed from 76% to 47% in 12-12.1 min; the liquid B is linearly changed from 47 percent to 95 percent in 12.1-15.1min and is maintained at 95 percent in 15.1-22 min;
the solution A in the step S3 is 10mM ammonium acetate water solution, pH 9.2; the solution B is 10mM ammonium acetate water solution dissolved in 85% acetonitrile water solution and has a pH value of 9.2.
Step S4 mass spectrometry, wherein the sample subjected to chromatographic separation in step S3 directly enters mass spectrometry, and mass spectrometry conditions are that an ESI source is adopted, the ion source temperature: at 450 ℃; atomization air pressure: 45, a first step of; auxiliary air pressure: 45, a first step of; air curtain air: 30, of a nitrogen-containing gas; the spraying voltage is-4500V, and the multi-reaction monitoring mode is adopted for detection;
the metabolite quantitative ion pair and the metabolite qualitative ion pair of the mass spectrometry and the corresponding collision energy information are as follows:
an ion pair with good specificity is selected as a quantitative ion pair, a qualitative ion pair is used for assisting, and a TIC (time induced degradation) map of mass spectrometry is shown in FIG. 2.
The following further analysis is performed on the detection result of this embodiment to verify the feasibility of the method provided by the present invention: and establishing a standard curve by adopting an external standard quantitative method and taking the concentration of 20 energy metabolism standard substances as an X axis and the peak area value of the standard substance as a y axis. The concentration of each test substance in mouse heart tissue was calculated from 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 1 below.
TABLE 1 Standard Curve Table
The 20 energy metabolites have good linear relation in respective concentration linear ranges, meet the quantitative requirement, and can accurately quantify the content of the 20 important energy metabolites in the heart tissue of the mouse 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 (6)
1. The tissue energy metabolism substance analysis method based on the UPLC-MSMS is characterized by comprising the following steps:
s1, constructing a standard curve, respectively weighing energy metabolite standard substances, preparing an energy metabolite standard substance mixed solution with a series of gradient concentrations by using 50% acetonitrile aqueous solution, and directly performing the steps S3 and S4 on the standard substance mixed solution to obtain the standard curve;
step S2, preparing a sample, namely taking a tissue sample to be detected, adding water to prepare a tissue homogenate, adding a methanol/acetonitrile mixed solution, carrying out vortex mixing, carrying out ultrasonic treatment in ice bath, incubating precipitated protein, carrying out high-speed centrifugation to obtain a supernatant, and carrying out vacuum drying for later use;
and step S3, carrying out liquid chromatographic analysis, namely, redissolving the dried sample obtained in the step S2 by using an acetonitrile aqueous solution, centrifuging and taking the supernatant to carry out liquid chromatographic analysis, wherein the chromatographic conditions are as follows:
injecting sample at 4 deg.C, column temperature 45 deg.C, and flow rate 300 μ L/min; the liquid phase gradient was as follows: the liquid B is linearly changed from 95% to 76% in 0-12 min; the liquid B is linearly changed from 76% to 47% in 12-12.1 min; the liquid B is linearly changed from 47 percent to 95 percent in 12.1-15.1min and is maintained at 95 percent in 15.1-22 min;
step S4 mass spectrometry, wherein the sample subjected to chromatographic separation in step S3 directly enters mass spectrometry, and mass spectrometry conditions are that an ESI source is adopted, the ion source temperature: at 450 ℃; atomization air pressure: 45, a first step of; auxiliary air pressure: 45, a first step of; air curtain air: 30, of a nitrogen-containing gas; the spraying voltage is-4500V, and the detection is carried out by adopting a multi-reaction monitoring mode.
2. The UPLC-MSMS-based tissue energy metabolism assay method according to claim 1, wherein the concentration of the series of gradients in step S1 is in the range of 1-50000ng/mL, and the energy metabolite standards comprise aconitic acid, adenosine diphosphate, α -ketoglutaric acid, adenosine monophosphate, adenosine triphosphate, citric acid, cyclic adenosine monophosphate, flavin mononucleotide, fumaric acid, guanosine 5 '-diphosphate, guanosine 5' -guanosine triphosphate, guanosine isocitrate, malic acid, nicotinamide adenine dinucleotide phosphate, phosphoenolpyruvate, pyruvic acid, succinic acid, and triphenyl phosphate.
3. The method for analyzing tissue energy metabolites based on UPLC-MSMS according to claim 1, wherein the water and methanol/acetonitrile mixture of step S2 is pre-cooled to a temperature of 4 ℃, the water is ultra-pure water, and the methanol/acetonitrile mixture is a mixture of methanol and acetonitrile in a volume ratio of 1: 1, the ultrasonic treatment is 350W treatment for 20min, and the incubation of the precipitated protein is carried out for 1h at the temperature of minus 20 ℃.
4. The method for analyzing tissue energy metabolism substance according to claim 1, wherein the centrifugation in steps S2 and S3 is 14000rcf at 4 ℃ for 15 min.
5. The UPLC-MSMS-based tissue energy metabolism assay method according to claim 1, wherein the dried sample is reconstituted with 50% acetonitrile in step S3; the solution A in the step S3 is 10mM ammonium acetate water solution, pH 9.2; the solution B is 10mM ammonium acetate water solution dissolved in 85% acetonitrile water solution and has a pH value of 9.2.
6. The UPLC-MSMS-based tissue energy metabolism analysis method according to claim 1, wherein the metabolite quantitative ion pairs, the qualitative ion pairs, and the corresponding collision energy information of the mass spectrometry analysis in step S4 are as follows:
And selecting an ion pair with good specificity as a quantitative ion pair and assisting with a qualitative ion pair.
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