A method for measuring the mitochondrial energy metabolome

Abstract

The present invention discloses a method for determining the mitochondrial energy metabolome. The method constructs the mitochondrial energy metabolome based on the relevant pathways of mitochondrial energy metabolism, adopts N,N-dimethyl-p-phenylenediamine (DMPD) as a derivatization reagent for target metabolites, and establishes a quantitative analysis method based on UPLC-MS / MS. The mitochondrial energy metabolome includes relevant metabolites in tricarboxylic acid cycle, glycolysis, amino acid metabolism, fatty acid metabolism and nucleotide metabolism. The present invention reacts with a derivatization reagent and a metabolite, has high derivatization efficiency and is easy to operate; a hydrophobic group is introduced after derivatization, thereby improving the retention of the metabolite on a reverse phase chromatographic column; the nitrogen element carried by the derivatization product is easily protonated in the positive ESI mode, thereby improving the detection sensitivity in the mass spectrometer.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and specifically relates to a method for determining mitochondrial energy metabolome by combining derivatization with ultra-high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Mitochondria are important bioenergetics and biosynthetic organelles that are essential for maintaining normal cell function and human health. Mitochondria are the hub of metabolic reactions and are associated with multiple metabolic pathways, including sugar metabolism (tricarboxylic acid cycle and glycolysis), amino acid metabolism, fatty acid metabolism, and nucleotide metabolism. Mitochondrial dysfunction can cause a variety of diseases, and an important hallmark of malignant tumors is metabolic reprogramming, which is driven by mitochondria through a variety of mechanisms. Therefore, in order to better study the relationship between mitochondrial metabolism and various diseases, it is urgent to establish a highly sensitive analytical method that can simultaneously detect multiple mitochondrial metabolites.

[0003] The advantages of LC-MS / MS such as high selectivity, specificity and sensitivity have made it the most important detection method in metabolomics. Most mitochondrial metabolites are water-soluble metabolites, including small molecule organic acids, amino acids and nucleotides. These metabolites are highly polar and have poor retention on reversed-phase columns. The methods currently used are: adding ion pair reagents to improve retention, but this method is prone to contaminating the mass spectrometer; using hydrophilic interaction chromatography (HILIC), which has better retention for polar compounds, but the method optimization is complex; derivatization methods can also be used to introduce hydrophobic groups to improve retention, and mass spectrometry sensitization groups can also be introduced to improve mass spectrometry response.

[0004] In summary, it is of great value to develop a derivatization-based LC-MS method that can detect mitochondrial metabolites simply, efficiently, selectively, and sensitively. Summary of the invention

[0005] The purpose of the present invention is to provide a method for determining the mitochondrial energy metabolome by combining derivatization with LC-MS. By adopting a derivatization reagent, it is possible to simultaneously detect multiple mitochondrial metabolites and improve their chromatographic retention and mass spectrometry response.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for measuring mitochondrial energy metabolome, comprising the following steps:

[0008] Step 1, derivatization method: take a metabolic extract of cells or tissues, then add 10-50mM N,N-dimethyl-p-phenylenediamine solution, 5-30mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and 0.5-10mM imidazole solution in sequence, mix well and react in a 30-60°C water bath for 10-120min;

[0009] Step 2, purification of the derivatized product: add a mixed solution of water and ethyl acetate to the reaction solution for extraction, centrifuge at 13000 rpm, 4°C for 5 min, draw the water layer, blow nitrogen to dryness, re-dissolve in 3% acetonitrile aqueous solution, and add internal standard solution;

[0010] Step 3, using ultra-high performance liquid chromatography-tandem mass spectrometry to inject and detect the solution obtained in step 2, and calculating the content of each metabolite through a standard curve;

[0011] The detection conditions of the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows:

[0012] Chromatographic column: CORTECS UPLC T3 Column, specification: 2.1×150mm, 1.6μm; column temperature: 45℃; injection volume: 2μL; flow rate: 0.2mL / min; mobile phase composition: mobile phase A: 10mM ammonium formate aqueous solution; mobile phase B: acetonitrile; gradient elution was used, and the elution conditions are as follows:

[0013]

[0014] Mass spectrometry conditions: multiple reaction monitoring mode, ESI ion source, positive electrospray ion mode operation, the parameters used are as follows: drying gas value, 110; nebulizer gas value, 180; electrospray voltage, 5500 V; ion source temperature, 400 °C.

[0015] Further, the mitochondrial metabolites include succinate, α-ketoglutarate, malate, fumarate, citric acid, 2-hydroxyglutarate, oxaloacetate, aconitic acid, lactate, creatine, aspartic acid, asparagine, alanine, proline, adenosine monophosphate, adenosine triphosphate, cytosine nucleotide, uracil nucleotide, carnitine, acetylcarnitine, propionylcarnitine, betaine, and dihydroorotic acid.

[0016] Further, the concentration of the N,N-dimethyl-p-phenylenediamine solution is 40 mM, the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution is 10 mM, and the concentration of imidazole is 1 mM.

[0017] In one embodiment of the present invention, the volume ratio of the metabolic extract of cells or tissues, the N,N-dimethyl-p-phenylenediamine solution, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and the imidazole solution is 1:1:1:2.

[0018] Furthermore, after mixing in step 1, the mixture is reacted in a 50° C. water bath for 90 minutes.

[0019] The present invention has the following advantages and beneficial effects:

[0020] (1) The quantitative method of the present invention covers a variety of metabolites related to mitochondrial energy metabolism, which can further study the relationship between mitochondrial metabolic changes and various diseases.

[0021] (2) The present invention is the first to use N,N-dimethyl-p-phenylenediamine as a derivatization reagent to derivatize the carboxyl sites of mitochondrial metabolites. The derivatization reaction conditions are simple, mild, rapid, and environmentally friendly, and the derivatization steps are simple.

[0022] (3) The quantitative method of the present invention is based on a highly sensitive triple quadrupole mass spectrometer, which has a fast analysis speed and a low quantitative limit, and can meet the detection of relevant metabolites in the sample to the greatest extent.

[0023] (4) This method introduces hydrophobic groups into the derivatized product, which improves the retention of water-soluble metabolites with high polarity on the reversed-phase chromatographic column. The introduced groups also increase the sensitivity of metabolites in mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The ion fragmentation patterns of derivatized succinic acid, lactic acid, alanine and monoadenosine phosphate, where a is succinic acid, b is lactic acid, c is alanine, and d is monoadenosine phosphate.

[0025] Figure 2-Figure 6 These are the optimization results of derivatization temperature, derivatization time, DMPD concentration, EDC concentration and imidazole buffer concentration.

[0026] Figure 7 This is the extracted ion current chromatogram of the multiple reaction monitoring of the mitochondrial energy metabolism standard after derivatization. Among them: 1-adenosine triphosphate, 2-citrate, 3-asparagine, 4-uracil nucleotide, 5-cytosine nucleotide, 6-adenosine monophosphate, 7-aspartic acid, 8-malic acid, 9-cis-aconitic acid, 10-succinic acid, 11-2-hydroxyglutaric acid, 12-alanine, 13-fumaric acid, 14-betaine, 15-carnitine, 16-creatine, 17-α-ketoglutaric acid, 18-dihydroorotic acid, 19-acetylcarnitine, 20-lactic acid, 21-propionylcarnitine, 22-proline, 23-oxaloacetic acid.

[0027] Figure 8 The results show the changes in metabolite content of mitochondrial energy metabolome in HCT 116 cells after rotenone treatment. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but it should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods and reagents without specifying the specific conditions in the examples are all in accordance with the conventional conditions in the art.

[0029] Experimental materials and instruments

[0030] (1) Instruments: high-speed centrifuge, constant temperature water bath, Qsight LX50 ultra-high performance liquid chromatography system, Qsight220 triple quadrupole mass spectrometer (PerkinElmer).

[0031] (2) Reagents and consumables: All reagents used in the following examples unless otherwise specified were commercial reagents, among which N,N-dimethyl-p-phenylenediamine (DMPD) and imidazole were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., N-methyl-p-phenylenediamine (MPD) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., acetonitrile and methanol (chromatographic grade, Merck), and mass spectrometry grade ammonium formate (Sigma-Aldrich).

[0032] Example 1

[0033] 1. The derivatization method and product purification method of 23 mitochondrial energy metabolite standards are as follows:

[0034] (1) Preparation of standard solution: Accurately weigh each mitochondrial energy metabolism metabolite standard, add 10% methanol water to dissolve, prepare a 10mM single standard stock solution, and store in a -20℃ refrigerator. Take 10μL of each standard stock solution and mix them, add 10% methanol water to prepare a 200μM mixed standard solution.

[0035] (2) Preparation of derivatization reagents: DMPD solution: weigh DMPD and dissolve it in chromatographic grade acetonitrile to prepare a 200 mM solution; EDC solution: weigh EDC·HCl and dissolve it in water to prepare a 50 mM solution; weigh imidazole and dissolve it in water to prepare an imidazole buffer solution with a concentration of 2.5 mM.

[0036] (3) Derivatization reaction: Take 10 μL of the mixed standard solution in a 1.5 mL EP tube, then add 10 μL of EDC solution, 10 μL of DMPD solution and 20 μL of imidazole solution in sequence, vortex mix, and react in a 50°C water bath for 1.5 hours to obtain a derivatization product solution.

[0037] Take 10 μL of the mixed standard solution in a 1.5 mL EP tube, then add 10 μL of EDC solution, 10 μL of MPD solution and 20 μL of imidazole solution in sequence, vortex mix, and react in a 50 °C water bath for 1.5 hours to obtain an internal standard solution.

[0038] (4) Purification of derivatized product: 150 μL of water and 150 μL of ethyl acetate were added to the derivatized product solution, vortexed, extracted for 1 min, centrifuged at 13,000 rpm, 4°C for 5 min, aspirated the aqueous layer, purged with nitrogen to dryness, re-dissolved in 3% acetonitrile aqueous solution, and added the internal standard solution to the derivatized product solution.

[0039] 2. Optimization of Derivatization Reaction Conditions

[0040] (1) Optimization of derivatization temperature

[0041] In the present invention, the derivatization reaction between the mitochondrial energy metabolism group standard solution and DMPD is tested in a 4° C. refrigerator, a 30° C. water bath, a 50° C. water bath and a 60° C. water bath.

[0042] The specific implementation plan is as follows: take 10 μL of the mixed standard solution into a 1.5 mL EP tube, then add 10 μL of LEDC solution, 10 μL of DMPD solution and 20 μL of imidazole solution in sequence, vortex mix, and react in a 4°C refrigerator, 30°C, 50°C and 60°C water bath for 1.5 hours, respectively. After the obtained product is purified, the internal standard solution is added, and the sample is detected by ultra performance liquid chromatography-triple quadrupole mass spectrometry.

[0043] (2) Optimization of derivatization time

[0044] The present invention tests the derivatization reaction of the mitochondrial energy metabolism group standard solution with DMPD for 10 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes respectively.

[0045] The specific implementation plan is as follows: take 10 μL of the mixed standard solution into a 1.5 mL EP tube, then add 10 μL of DMPD solution, 10 μL of EDC solution and 20 μL of imidazole solution in sequence, vortex mix, and react in a 50°C water bath for 10 min, 30 min, 60 min, 90 min and 120 min, respectively. After the obtained product is purified, the internal standard solution is added, and the sample is detected by ultra performance liquid chromatography-triple quadrupole mass spectrometry.

[0046] (3) Optimization of DMPD, EDC and imidazole concentrations

[0047] The present invention reacts a mitochondrial energy metabolism group standard solution with different concentrations of DMPD (10mM, 20mM, 30mM, 40mM, 50mM), different concentrations of EDC (5mM, 10mM, 20mM, 30mM) and different concentrations of imidazole buffer (0.5mM, 1mM, 2mM, 5mM, 10mM).

[0048] The specific implementation plan is as follows: take 10 μL of the mixed standard solution into a 1.5 mL EP tube, then add 10 μL of DMPD solution, 10 μL of EDC solution and 20 μL of imidazole solution in sequence, vortex mix, and react in a 50°C water bath for 1.5 hours. After the obtained product is purified, the internal standard solution is added, and the sample is detected by ultra performance liquid chromatography-triple quadrupole mass spectrometry.

[0049] like Figure 2-Figure 6 As shown, the optimal derivatization temperature of the derivatization reaction is 50°C, the optimal derivatization time is 90 min, the optimal concentration of DMPD is 40 mM, the optimal concentration of EDC is 10 mM, and the optimal concentration of imidazole is 1 mM.

[0050] 3. Inject the derivatized product prepared in (a) for ultra-high performance liquid chromatography-triple quadrupole mass spectrometry analysis

[0051] (1) Precursor ion and product ion scanning analysis: Figure 1 a, 1b, 1c, and 1d are the product ion scans of succinate, lactate, alanine, and adenosine monophosphate, respectively. Figure 2 The results of mass spectrometry analysis showed that in the positive ion mode, the derivative products could form [M+H] after obtaining a H + The parent ion peak of Figure 1 a. Figure 1 b. Figure 1 c Product ion mass spectrum. For metabolites containing carboxyl groups, fragment ions that lose a CH3 are easily generated, and their derivative products all produce a characteristic fragment ion peak of m / z 122.1, with the same fragmentation mode and good response. Figure 1 d It can be seen that the metabolites containing phosphate groups have their derivative products producing characteristic fragment ions of m / z 216.0.

[0052] The parameters of the mass spectrometry multiple reaction monitoring (MRM) mode were optimized, and the results are shown in Table 1.

[0053] Table 1 Mass spectrometry detection parameters of the derivatized product and internal standard provided in Example 1

[0054]

[0055]

[0056] (2) Liquid quality conditions:

[0057] Chromatographic conditions: Chromatographic column: CORTECS UPLC T3 Column (2.1×150mm, 1.6μm); column temperature: 45°C; injection volume: 2μL; flow rate: 0.2mL / min; mobile phase composition: mobile phase A: ultrapure water (0.1% formic acid); mobile phase B: acetonitrile; gradient elution was used, see Table 3 for details.

[0058] Mass spectrometry conditions: multiple reaction monitoring mode, ESI ion source, positive electrospray ion mode operation, the parameters used are as follows: drying gas value, 110; nebulizer gas value, 180; electrospray voltage, 5500 V; ion source temperature, 400 °C.

[0059] Table 3 Mobile phase gradient elution parameters

[0060]

[0061] (3) Determination of standard curve:

[0062] The mixed standard solution was accurately drawn, and a series of standard working solutions were prepared by diluting with 10% methanol water. Each solution was derivatized separately, and the obtained product was purified, and then the internal standard was added. The sample was detected by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The ratio of the area of ​​the chromatographic peak of each compound to the area of ​​the chromatographic peak of the internal standard was subjected to linear regression analysis with the concentration of each compound. The linear regression equation and correlation coefficient of each compound are shown in Table 4.

[0063] Table 4 Standard curves, correlation coefficients and linear ranges of mitochondrial energy metabolome compounds

[0064]

[0065] Example 2

[0066] The effect of rotenone administration on the mitochondrial energy metabolome of colon cancer cell HCT 116 was determined as follows:

[0067] 1. Cellular drug delivery and metabolite extraction

[0068] (1) Cell culture and administration: HCT 116 cells were cultured in high-glucose DMEM complete medium containing 10% fetal bovine serum. After the cells were inoculated into culture flasks, they were cultured in a cell culture incubator at 37°C and 5% CO2. When the cells reached 80% confluence, they were treated with a rotenone solution (concentration of 100 nM) dissolved in dimethyl sulfoxide (DMSO) and a 1% DMSO vehicle control solution for 12 hours.

[0069] (2) Extraction of cell metabolites: discard the culture medium, wash once with 2 mL PBS buffer, transfer the cells to a 1.5 mL EP tube, centrifuge at 13,000 rpm, 4°C for 1 min, discard the supernatant, add 100 μL of -80°C precooled extraction solvent (methanol: acetonitrile: water = 5:3:2) to the precipitate, vortex vigorously for 3 min, centrifuge at 13,000 rpm, 4°C for 1 min, and aspirate the supernatant as the extracted metabolites.

[0070] 2. Derivatization reaction and sample injection detection of cell metabolites

[0071] Take 10 μL of extracted cell metabolites in a 1.5 mL EP tube, then add 10 μL DMPD solution, 10 μL LEDC solution and 20 μL imidazole solution in sequence, vortex mix, and react in a 50 ° C water bath for 1.5 hours. After purification, add the internal standard solution to the obtained product, centrifuge at 13000 rpm, 4 ° C, and centrifuge for 10 min. Take 80 μL of the supernatant and detect it with ultra performance liquid chromatography-triple quadrupole mass spectrometry.

[0072] like Figure 7 As shown in the results, rotenone administration has an effect on the mitochondrial energy metabolome. As an inhibitor of mitochondrial respiratory chain enzyme complex 1, rotenone significantly reduced the content of intracellular adenosine triphosphate after administration, and the content of tricarboxylic acid cycle-related metabolites such as α-ketoglutarate, malate, citric acid and fumarate also decreased.

[0073] The above-described embodiments are only preferred embodiments of the present invention, but not all feasible embodiments of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for measuring mitochondrial energy metabolism, characterized in that: The following steps are involved: Step 1, derivatization method: take the metabolic extract of cells or tissues, then add 10-50mM N, N-dimethyl-p-phenylenediamine solution, 5-30mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and 0.5-10mM imidazole solution in sequence, mix well and react in a 30-60°C water bath for 10-120min; The volume ratio of the metabolic extract of the cell or tissue, N,N-dimethyl-p-phenylenediamine solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and imidazole solution is 1:1:1:2; Step 2, purification of the derivatized product: add a mixed solution of water and ethyl acetate to the reaction solution for extraction, centrifuge at 13000 rpm, 4°C for 5 min, draw the water layer, blow nitrogen to dryness, re-dissolve in 3% acetonitrile aqueous solution, and add internal standard solution; Step 3, using ultra-high performance liquid chromatography-tandem mass spectrometry to inject and detect the solution obtained in step 2, and calculating the content of each metabolite through a standard curve; The detection conditions of the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows: Chromatographic column: CORTECS UPLC T3 Column, specifications: 2.1 × 150 mm, 1.6 μm; Column temperature: 45°C; injection volume: 2 μL; flow rate: 0.2 mL / min; mobile phase Composition: Mobile phase A: 10 mM ammonium formate aqueous solution; Mobile phase B: acetonitrile; Gradient elution was used, and the elution conditions were as follows: Mass spectrometry conditions: multiple reaction monitoring mode, ESI ion source, operated in positive electrospray ion mode, the parameters used were as follows: drying gas value, 110; nebulizer gas value, 180; electrospray voltage, 5500 V; ion source temperature, 400 °C.

2. The method according to claim 1, characterized in that: The mitochondrial energy metabolome includes succinate, α-ketoglutarate, malate, fumarate, citric acid, 2-hydroxyglutarate, oxaloacetate, aconitic acid, lactate, creatine, aspartic acid, asparagine, alanine, proline, adenosine monophosphate, adenosine triphosphate, cytosine nucleotide, uracil nucleotide, carnitine, acetylcarnitine, propionylcarnitine, betaine, and dihydroorotic acid.

3. The method according to claim 1, characterized in that: The concentration of the N, N-dimethyl-p-phenylenediamine solution was 40 mM, the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution was 10 mM, and the concentration of imidazole was 1 mM.

4. The method according to claim 1, characterized in that: After mixing in step 1, react in a 50°C water bath for 90 minutes.

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