A method for detecting the ability of mitochondria to take up pyruvate in solution

Abstract

The present invention relates to a method for detecting the ability of mitochondria to absorb pyruvate in a solution. 13 C-labeled pyruvate is detected using gas chromatography-mass spectrometry, with natural, unlabeled pyruvate serving as an internal standard to reduce instrumental and operational errors. This method overcomes the limitations of radioisotope handling, often requiring high qualifications, and boasts lower instrumental errors and higher detection sensitivity than traditional colorimetric methods. Therefore, this method can be used to monitor the activity of the mitochondrial pyruvate transporter MPC1 / 2.

Description

Technical Field

[0001] The present invention relates to a method for qualitatively and quantitatively detecting the pyruvate uptake capacity of cell mitochondria using the gas chromatography-mass spectrometry technique described in the present application, which is used to characterize the activity of MPC (Mitochondrial pyruvate carrier) and belongs to the field of biotechnology. Background Art

[0002] Pyruvate is a key metabolite connecting cellular glycolysis and the tricarboxylic acid cycle. Numerous studies have shown that pyruvate metabolism is inextricably linked to the development and progression of cancer. Whether pyruvate can enter the mitochondria and enter the tricarboxylic acid cycle plays a crucial role in this process. A key characteristic of tumor cell metabolism is that under aerobic conditions, cells take up more glucose, producing large amounts of lactate, a phenomenon known as the Warburg effect. Glycolysis produces two pyruvate molecules per glucose molecule. Pyruvate can then be metabolized through two main pathways: transport through the MPC into the inner mitochondrial membrane and participate in the tricarboxylic acid cycle, or through LDH-catalyzed production of lactate. Furthermore, studies have reported that the absence or inhibition of MPC protein activity in mouse models increases the incidence of colorectal cancer. This is primarily due to the inability of the pyruvate to enter the mitochondria, where it is catalyzed to produce lactate, leading to enhanced glycolysis, increased cell stemness, and the development of cancer.

[0003] Currently, the proteins that control pyruvate transport in mammals are MPC1 / 2. Numerous studies have shown that MPC activity affects the glycolytic capacity of tumor cells, thereby influencing the development and progression of cancer. Therefore, a stable and reliable experimental technique is needed to measure MPC activity and characterize the metabolic profile of tumor cells.

[0004] There are two main methods for detecting the ability of MPC to transport pyruvate. 14 Mitochondria are incubated with a pyruvate reaction solution labeled with a C radioisotope, and the mitochondrial radioactivity is measured using a scintillometer. Pyruvate uptake capacity is characterized by mitochondrial radioactivity. This method has the advantages of short operation time and high sensitivity. However, due to the safety risks associated with the use of radioisotopes, the procedure must be performed at research institutions with the necessary qualifications to operate and purchase radioisotopes, significantly limiting its application. A second method is the pyruvate colorimetric method, which measures the remaining pyruvate content in the supernatant after pyruvate uptake. The difference between the remaining pyruvate content and the initial pyruvate content represents the pyruvate uptake level. The colorimetric method has the advantages of simplicity and speed. However, the principle of the pyruvate colorimetric method is to measure the amount of acetyl-CoA generated by the pyruvate reaction, which is easily affected by mitochondrial metabolism and residual acetyl-CoA. Furthermore, the instrument error ranges from 1% to 5%, resulting in low resolution. Given that mitochondrial pyruvate uptake in vitro is relatively small, colorimetric methods often produce inaccurate measurements and require a large number of mitochondria, increasing experimental costs.

[0005] Gas phase mass spectrometry tandem technology has the characteristics of high safety, high detection sensitivity and high resolution. 13 C-labeled pyruvate and natural unlabeled pyruvate as an internal standard. Accurate quantification is possible. 13 The C-labeled pyruvate content ultimately provides the accurate mitochondrial pyruvate uptake.

[0006] Currently, there is no report on the application of the stable isotope combined with gas chromatography-mass spectrometry mentioned in the present invention in detecting the mitochondrial pyruvate uptake capacity. Summary of the Invention

[0007] This invention involves studying the activity of the mitochondrial pyruvate transporter. One objective is to investigate the ability of tumor cell mitochondria to utilize pyruvate by measuring mitochondrial pyruvate transport capacity, thereby further exploring the role of this process in tumor development and progression. A second objective is to provide a highly sensitive and low-error method for measuring mitochondrial pyruvate uptake capacity for research institutions lacking radioisotope handling qualifications.

[0008] Experiments have shown that the detection method described in the present invention can accurately detect the mitochondrial pyruvate uptake capacity and can therefore be used to detect the activity of MPCs.

[0009] Pyruvate uptake was performed using 13 C fully labeled pyruvate, gas chromatography-mass spectrometry as the detection means, and natural unlabeled pyruvate as the internal standard to reduce instrument and operation errors.

[0010] This method overcomes the limitations of radioisotope handling, often requiring high qualifications, and boasts lower instrumental error and higher detection sensitivity than traditional colorimetric methods. Therefore, it can be used to study the activity of the mitochondrial pyruvate transporter MPC1 / 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 . 13 C3 Pyruvate was used to detect the pyruvate uptake capacity of mitochondria in 293T cells overexpressing MPC1 / 2.

[0012] Figure 2 . 13 C3 Pyruvate was used to detect the mitochondrial pyruvate uptake capacity of MDA-MB-231 cells overexpressing MPC1 / 2. DETAILED DESCRIPTION

[0013] The present invention will now be further described with reference to examples, which are intended to illustrate the present invention but not to limit it.

[0014] The stable isotope label used in the present invention for pyruvate uptake is [U-13 [C3] Pyruvate, an unlabeled pyruvate, is used as an internal standard to reduce instrument and operator errors. Reaction Solution A, described herein, comprises: 120 mM KCl, 5 mM KH2PO4, 1 mM EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid), 3 mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), pH 7.4.

[0015] The components of reaction solution B are: 120mM KCl, 5mM KH2PO4, 1mM EGTA, 3mM HEPES pH 6.8, 0.075mM Malate (sodium malate), 0.15mM 13 C3Pyruvate.

[0016] Extraction agent C is a 0.05 mM methanol solution of Pyruvate (sodium pyruvate).

[0017] Example 1

[0018] 293T cells were transfected with a plasmid containing the MPC1 / 2 gene (MPC1 and MPC2 protein overexpression plasmids were cloned and inserted into the pBobi vector using enzyme ligation. The restriction endonucleases used were BamHI and Xho I, the mass ratio of MPC1 and MPC2 was one to one, 10 ug of the mixed plasmid was transfected into each 10 cm culture dish, and the control group was transfected with the pBobi empty vector plasmid) and cultured in DMEM medium containing 10% FBS (fetal bovine serum) in an incubator at 37°C in an atmosphere of 5% CO2 by volume. 48 hours after plasmid transfection, the cells were washed twice with PBS (phosphate buffered saline), and the cells were scraped with PBS and collected into a 50 ml tube. Mitochondria were extracted (mitochondria followed the method used in the literature [Frezza, C., S. Cipolat, and L. Scorrano, Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc, 2007. 2 (2): p. 287-95] and the mitochondrial concentration was adjusted to a consistent level.

[0019] Mitochondrial pyruvate uptake test steps:

[0020] 1. Resuspend mitochondria in 100 μl of reaction solution A (final concentration 10 mg / ml) gently.

[0021] 2. Add 200 μl of reaction solution B and incubate at room temperature for 10 minutes, mixing every 30 seconds.

[0022] 3. Centrifuge at 10,000g, 4°C for 1 minute.

[0023] 4. Pipette 80 μl of the supernatant after the reaction into a 1.5 ml EP tube. Add 400 μl of extraction agent C to the EP tube in advance. Set up three parallel tubes.

[0024] 5. Vortex for 15 seconds and centrifuge at 10,000 g, 4°C for 10 minutes.

[0025] 6. Transfer 400 μl of sample supernatant to a new 1.5 ml EP tube and freeze-dry overnight at 4°C.

[0026] 7. Add 30 μl of methoxyamine pyridine solution (20 mg / ml, methoxyamine powder dissolved in pyridine solution) to the lyophilized sample, vortex for 15 seconds, and incubate in a 37°C water bath for 1 hour. Seal the EP tube with parafilm to prevent water ingress.

[0027] 8. Add 30 μl of MTBSTFA (purchased from Sigma, product number M-108) to the EP tube, vortex for 15 seconds, and place in a 55°C water bath for 2 hours.

[0028] 9. Centrifuge at 10,000 g for 15 minutes at room temperature, transfer the supernatant to the upper sample, and then perform gas chromatography-mass spectrometry analysis.

[0029] The pyruvate content obtained by mass spectrometry is defined as the ratio of M+3 pyruvate to M+0 pyruvate (M+3 means that all three Cs of pyruvate in the mass spectrometry result are 13 C substitution, the corresponding M+0 means that all three Cs of pyruvate are 12 C) Mitochondrial pyruvate uptake is the difference between the initial pyruvate amount and the amount of pyruvate remaining in the supernatant. Figure 1 As shown, overexpression of the pyruvate transporter MPC1 / 2 as a positive control significantly increased mitochondrial pyruvate uptake with minimal measurement error.

[0030] Example 2

[0031] MDA-MB-231 cells were infected with lentivirus (a third-generation lentivirus packaging system, i.e., a mixture of pMDL, pVSVg, and pREV in a mass ratio of 5:3:2) carrying MPC1 / 2 (pBobi vector of MPC1 and MPC2) (virus packaging followed the addgene virus packaging experimental process). After 48 hours of virus infection, Puromycin (puromycin) was added at a final concentration of 2ug / mL for screening for 48 hours to establish a stable cell line that stably expresses MPC1 / 2. The cells were expanded and cultured in a 15cm dish, with 15 dishes each for the control group and the overexpression experimental group. When the cell density reached more than 90%, the cells were scraped off to extract mitochondria, and the two groups of mitochondria were adjusted to the same number. Process according to the operating steps in Example 1. Three were parallel. Gas chromatography-mass spectrometry was then performed. The results are shown as follows. Figure 2 Therefore, overexpression of MPC1 / 2 significantly improves mitochondrial pyruvate transport capacity in 231 cells. The measurement is accurate and has a small error.

[0032] The above experiments show that the use of stable isotopes 13 C-labeled pyruvate combined with gas chromatography-mass spectrometry can accurately measure mitochondrial pyruvate uptake capacity. This method avoids the safety risks and qualification restrictions associated with the use of radioisotopes and offers higher accuracy and lower error than colorimetric methods. It provides a promising candidate for the study of mitochondrial pyruvate transport and holds great promise for both technology and commercialization.

Claims

1. A method for detecting the ability of mitochondria to take up pyruvate in a solution, characterized in that: The detection method based on stable isotope tracing combined with gas phase mass spectrometry technology, the stable isotope is 13 C-labeled pyruvate and natural unlabeled pyruvate were used as internal standards. The pyruvate content in the solution before and after incubation was detected by gas chromatography-mass spectrometry to obtain the pyruvate uptake by mitochondria in the solution.

2. The method according to claim 1, wherein: The pyruvic acid in the solution is 13 The concentration of C fully labeled pyruvate is 0.05 mM to 0.15 mM, and the concentration of native unlabeled pyruvate is 0.025 mM to 0.1 mM.

3. The method according to claim 1, wherein: The pyruvate uptake is the difference between the initial pyruvate reaction amount and the remaining supernatant amount after the reaction. The pyruvate measurement value is defined as 13 C is the ratio of the mass spectrum peak area of ​​fully labeled pyruvate to the mass spectrum peak area of ​​native unlabeled pyruvate.

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