Combined metabolic marker for mitochondrial purity evaluation and detection kit

Abstract

The invention relates to a combined metabolic marker for mitochondrial purity evaluation and a detection kit. The marker comprises phosphatidylcholine PC 34: 0 (16: 0 / 18: 0) and sphingomyelin SM34: 1. O (d18: 1, 2O / 16: 0), sphingomyelin SM 44: 2; 20 (18: 1, 20 / 24: 1) and hexose ceramide HexCer42: 2; 2O (d18: 1; 2O / 24: 1), a new application for determining the purity of the mitochondria extracted by the mitochondria extraction method, and a kit. According to the method, the relative concentrations of four lipid metabolites in a mitochondrial sample are detected, a combined marker variable P and a determined intercept point value are calculated based on a binary logistic regression equation, and the mitochondrial purity extracted by the method is judged. The kit can realize high-sensitivity and high-efficiency detection of the four small molecule metabolites involved in the invention, and has the characteristics of low detection cost and good repeatability.

Description

Technical Field

[0001] The present invention relates to the fields of analytical chemistry, cell biology and molecular biology, and in particular to a combined metabolic marker and a detection kit for evaluating mitochondrial purity. Background Art

[0002] Mitochondria are a key organelle that is widely present in eukaryotic cells. As the "power plant" of cells, mitochondria release the energy stored in food decomposition products through respiration, providing the necessary energy support for various metabolic activities of cells. The mitochondrial tricarboxylic acid cycle (TCA cycle) is one of the key pathways for cell energy production and plays an important role in regulating metabolic activities in the body. In the human body, mitochondria are abundant in muscle cells and organs with high energy demands, such as the liver, heart, and brain. In addition to participating in energy metabolism, mitochondria also play an important role in many metabolic processes in the body, involving cell signaling, calcium ion buffering, reactive oxygen species (ROS) metabolism, cell apoptosis, fatty acid metabolism, and pyrimidine biosynthesis.

[0003] Mitochondrial dysfunction and mitochondrial defects are closely related to life and health. Studies have shown that mitochondria not only play an important role in the occurrence and development of many diseases such as tumors, cardiovascular diseases, neurodegenerative diseases, diabetes, and immune diseases, but also their abnormal activities are closely related to the progression of these diseases. For example, in Alzheimer's disease and other neurodegenerative diseases, the increase in Drp1 levels causes excessive mitochondrial fission, leading to mitochondrial dysfunction and neuronal damage; mitochondrial dysfunction also plays a key role in metabolic diseases such as insulin deficiency or insulin resistance such as type 2 diabetes; mitochondrial dysfunction in liver tissue is also related to the onset of various liver diseases such as fatty liver, hepatitis, and liver cancer.

[0004] Studies have shown that there are significant differences between the metabolic profiles of whole cell components and those at the subcellular level. In the study of mitochondrial subcellular metabolism, contamination from other organelles may not only lead to erroneous research conclusions, but also affect the comparability of data. Different extraction techniques will lead to differences in the purity of the obtained mitochondria. In order to ensure the rigor and accuracy of the study, it is crucial that the purity of the mitochondria used for the study matches the purpose of the study. Therefore, it is of great significance to evaluate the purity of mitochondria extracted by each purification method at the subcellular level for related mitochondrial research. Differential centrifugation (DC) and ultracentrifuge method (UC) are currently the two classic mitochondrial extraction methods. The principle of differential centrifugation (DC) is based on the different densities of different organelles at the subcellular level, and the purpose of separating or purifying mitochondria is achieved by setting different centrifugation conditions. Ultracentrifugation (UC) is a method based on differential centrifugation (DC) by setting solutions with different density gradients to further purify mitochondria by ultracentrifugation. Since grinding may cause damage to mitochondria, in order to reduce the damage to mitochondria, the column separation method (CO) is used as a new method for mitochondrial extraction. It uses density liquid to swell cells and breaks cells by separation columns, thereby effectively avoiding the damage to mitochondria caused by grinding, and then obtains mitochondria through density liquid and different centrifugation conditions. Since the centrifugation step may precipitate other organelles outside mitochondria, magnetic-activated cell sorting (MACS) is also a new mitochondrial extraction method that avoids multiple centrifugation steps and can accurately obtain mitochondria with high purity by combining antigens and antibodies.

[0005] Lipidomics is an emerging discipline that systematically analyzes the overall lipid metabolic profile. By comparing the changes in lipid metabolic networks under different physiological states, key lipid biomarkers in metabolic regulation are identified to reveal the mechanism of action of lipids in life activities. As the main component of the membranes of various subcellular organelles, lipids are involved in regulating many important processes of life activities, such as energy storage, material transport, signal transduction, cell development, differentiation and apoptosis. In recent years, lipid markers have been widely used in the clinical diagnosis and research of major diseases such as food nutrition and toxicity evaluation, cancer, diabetes, and neurodegenerative diseases. In view of the structural functions of lipid metabolites and their metabolic regulatory effects, the present invention uses lipidomics technology and research methods based on ultra-high performance liquid chromatography-mass spectrometry to analyze the lipid metabolic profiles of samples extracted by different mitochondrial extraction methods. By screening markers, the present invention discriminates the purity of mitochondria extracted by these methods. The present invention proposes a new use of the combined use of lipid metabolites phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1; 2O (d18:1; 2O / 16:0), sphingomyelin SM 44:2; 2O (18:1; 2O / 24:1) and hexosylceramide HexCer42:2; 2O (d18:1; 2O / 24:1) to evaluate mitochondrial purity. Since a single metabolite may be affected by multiple factors and lipids have multiple roles in organisms, a combined metabolic marker composed of a few metabolites is screened out from purified mitochondria, and the "discrimination possibility" P value is calculated using a discriminant formula, which helps to improve the sensitivity and specificity of the discrimination. At present, there is no report on the combined use of these four lipid metabolites for the study of discriminating mitochondrial purity. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a combined metabolic marker and detection kit for mitochondrial purity evaluation, which is specifically achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a combined metabolic marker for evaluating mitochondrial purity, which marker includes: phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1; 2O (d18:1; 2O / 16:0), sphingomyelin SM 44:2; 2O (18:1; 2O / 24:1) and hexosylceramide HexCer42:2; 2O (d18:1; 2O / 24:1).

[0008] The second aspect of the present invention provides the use of combined metabolic markers in evaluating mitochondrial purity.

[0009] The third aspect of the present invention provides a method for determining the purity of mitochondria in a mitochondrial extraction method using a combined metabolic marker, using a combined marker variable P for determination:

[0010] P=1 / (1+e^(-42.608a+5.583b-7.591c+7.343d+11.075))

[0011] Among them, a is the relative content of phosphatidylcholine PC 34:0 (16:0 / 18:0), b is the relative content of sphingomyelin SM34:1;2O (d18:1;2O / 16:0), c is the relative content of sphingomyelin SM44:2;2O (18:1;2O / 24:1), and d is the relative content of hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1). If P < 0.5, it is judged that the purity of mitochondria obtained by this method is higher than that of classical differential centrifugation (DC) and comparable to that of classical ultracentrifugation (UC). If P > 0.5, it is judged that the purity of mitochondria obtained by this method is lower than that of classical ultracentrifugation (UC) and comparable to that of classical differential centrifugation (DC).

[0012] The fourth aspect of the present invention provides a kit for detecting combined metabolic markers, comprising: standard substances: ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0) and sphingomyelin SM 30:1;2 (d18:1; / 12:0);

[0013] Pretreatment extract: A methanol solution containing internal standards of 1.27 μg / mL ceramide Cer 35:1;2 (d18:1 / 17:0), 2.17 μg / mL phosphatidylcholine PC 38:0 (19:0 / 19:0), and 1.38 μg / mL sphingomyelin SM 30:1;2 (d18:1; / 12:0). In positive ion detection mode, ceramide Cer 35:1;2 (d18:1 / 17:0) is used to calibrate hexosylceramide, sphingomyelin SM 30:1;2 (d18:1; / 12:0) is used to calibrate sphingomyelin, and phosphatidylcholine PC 38:0 (19:0 / 19:0) is used to calibrate phosphatidylcholine in negative ion detection mode.

[0014] Eluent: Mobile phase A is an acetonitrile / water solution containing 10 mM ammonium acetate (v / v=6:4), and mobile phase B is an isopropanol / acetonitrile solution containing 10 mM ammonium acetate (v / v=9:1).

[0015] A fifth aspect of the present invention provides a method for calculating a combined marker variable using a detection kit, comprising the following steps:

[0016] 1) Pretreatment of the mitochondrial samples: Methanol containing an internal standard was added to the samples from the mitochondria, followed by methyl tert-butyl ether and ultrapure water, and then lipid metabolites were extracted;

[0017] 2) separating and identifying the lipid metabolites of the mitochondrial test sample extracted after the treatment in step 1) based on ultra-high performance liquid chromatography-mass spectrometry;

[0018] 3) Use the ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0) and sphingomyelin SM 30:1;2 (d18:1; / 12:0) standards provided in the kit to assist in qualitative confirmation of the detected ions:

[0019] a) The ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0) and sphingomyelin SM 30:1;2 (d18:1; / 12:0) standards in the kit were analyzed by ultra-high performance liquid chromatography-mass spectrometry to determine the chromatographic retention time, measured mass-to-charge ratio, and secondary mass spectrometry characteristic ions of the standards. Combined with the LipidMaps database and the ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0) and sphingomyelin SM 30:1;2 (d18:1; / 12:0) standards in the kit, the secondary mass spectrometry bond breaking rules of ceramide, phosphatidylcholine and sphingomyelin were determined: in positive ion mode, the characteristic secondary of ceramide is the LCB chain, which is determined according to its dehydrated number and chain structure; sphingomyelin undergoes neutral loss, and a characteristic ion with an m / z of 184 can be obtained; in negative ion mode, the acyl chain of phosphatidylcholine breaks, generating characteristic fatty acid ions corresponding to different sites, and the corresponding characteristic ions are searched in the secondary mass spectrometry according to the m / z of the fatty acid ions;

[0020] b) In the tested mitochondrial samples, under a reasonable mass-to-charge ratio tolerance threshold, the chromatographic peaks of hexosylceramide HexCer42:2;2O (d18:1; 2O / 24:1) with a theoretical mass-to-charge ratio of 782.65039±5ppm, and the chromatographic peaks of sphingomyelin SM34:1;2O (d18:1; 2O / 16:0) and sphingomyelin SM44:2;2O (18:1; 2O / 24:1) with mass-to-charge ratios of 703.57489±5ppm and 841.7157±5ppm were extracted in the positive ion mode; and the chromatographic peak of phosphatidylcholine PC 34:0 (16:0 / 18:0) with a mass-to-charge ratio of 820.6073±5ppm was extracted in the negative ion mode;

[0021] c) Check whether the secondary mass spectrometry ions of the above four chromatographic peaks are consistent with the secondary mass spectrometry bond breaking rules of ceramide, sphingomyelin and phosphatidylcholine, and confirm the targets in combination with the chromatographic retention behavior: phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1; 2O (d18:1; 2O / 16:0), sphingomyelin SM 44:2; 2O (18:1; 2O / 24:1) and hexosylceramide HexCer42:2; 2O (d18:1; 2O / 24:1);

[0022] 4) For the target phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1; 2O (d18:1; 2O / 16:0), sphingomyelin SM 44:2; 2O (18:1; 2O / 24:1) and hexosylceramide HexCer42:2; 2O (d18:1; 2O / 24:1) in the identified mitochondrial test samples, their chromatographic peak intensities were corrected for the total elution peak area of ​​the mitochondrial samples to obtain the relative concentrations of the above four metabolites.

[0023] Phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1) and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1) were calculated by binary logistic regression to obtain the joint marker P.

[0024] The principle of the present invention is as follows:

[0025] 1) Using lipidomics analysis technology combined with ultra-high performance liquid chromatography-quadrupole and orbital trap hybrid Fourier transform ultra-high resolution mass spectrometry, lipidomics analysis was performed on mitochondrial lipid metabolic extracts extracted by classical mitochondrial purification methods, differential centrifugation (DC) and ultracentrifugation (UC) (discovery set, i.e., Example 1), to obtain qualitative and quantitative analysis results of lipid metabolites.

[0026] 2) Based on the comparison of mitochondrial lipid metabolism profiles obtained by classical differential centrifugation (DC) and ultracentrifugation (UC), differential lipid compounds are screened to discover potential markers. Specific approaches include:

[0027] i) Screening of typical differences in the metabolome: Based on univariate statistical significance (p<0.05&FDR<0.1) and multivariate analysis requirements (VIP>1), a set of typical differential metabolites was screened between differential centrifugation (DC) and ultracentrifugation (UC);

[0028] ii) Ratio screening: In the typical differential metabolite set, differential metabolites with a ratio of change greater than or equal to 10 (Ratio ≥ 10, Ratio ≤ 0.1) were screened.

[0029] iii) Analytical stability requirements: Based on more stringent data analysis quality requirements, potential markers were further screened with RSD < 5% in QC samples and mitochondrial metabolite samples extracted by differential centrifugation (DC);

[0030] ⅳ) Obtain the intersection of lipid metabolites of the above three data screening methods, and finally select the combination of phosphatidylcholine PC34:0 (16:0 / 18:0), sphingomyelin SM34:1; 2O (d18:1; 2O / 16:0), sphingomyelin SM 44:2; 2O (18:1; 2O / 24:1) and hexosylceramide HexCer42:2; 2O (d18:1; 2O / 24:1) as a joint marker, and analyze the P value of the joint marker. If P < 0.5, it is judged that the purity of mitochondria proposed by the purification method is high, and it is higher than the classical differential centrifugation (DC) method, and it is comparable to the classical ultracentrifugation (UC) method. If P > 0.5, it is judged that the purity of mitochondria proposed by the purification method is low, and it is lower than the classical ultracentrifugation (UC), and it is comparable to the classical differential centrifugation (DC).

[0031] 3) The mitochondria extracted by differential centrifugation (DC) and ultracentrifugation (UC) were analyzed by Western blot protein immunoblot to verify the above results.

[0032] 4) The effectiveness of the above-mentioned combined metabolic markers was further verified by comparing the column separation method (CO) with the magnetic bead method (MACS) (validation set, Example 2).

[0033] The present invention relates to a new application of lipid metabolites phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1) and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1) in mitochondrial samples, which are used in combination as markers for distinguishing mitochondrial purity. The present invention also relates to a kit for distinguishing mitochondrial purity. The kit can achieve high-sensitivity and high-efficiency detection of the four small molecule metabolites involved in the present invention, and has the characteristics of low detection cost and good repeatability. The combined use of the above four metabolites has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The structural formulas and secondary fragmentation characteristics of the four lipid markers are shown;

[0035] Figure 2 The contents of combined lipid markers in each group of samples (expressed as mean ± standard error) and ROC curve analysis;

[0036] Figure 3 Western Blot results of whole cell fractions and four mitochondrial extraction methods. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments to provide a better understanding of the present technical solution.

[0038] Example 1

[0039] 1. Mitochondrial Sample Collection

[0040] (1) Differential centrifugation (DC): When the cells are cultured to a coverage of 90% to 100% of the culture dish, the culture medium is aspirated and the cells are gently washed twice with 2 mL of PBS. Then, the cells are digested with trypsin at 37°C for two minutes and complete culture medium is added to terminate the digestion. The cells are gently blown to detach from the wall, and the suspension in the culture dish is collected into a centrifuge tube and centrifuged at 1000 g for 3 min. The supernatant is discarded, and the cells are resuspended and washed with 2 mL of KPBS and centrifuged at 1000 g for 3 min. The supernatant is then discarded and 1 mL of mitochondrial isolation reagent (Biyuntian, China) is added and resuspended. The resulting suspension is transferred to a Dounce homogenizer placed on ice in advance, and the cells are broken by grinding vertically up and down 35 times using a tight grinding pestle. 200 μL of homogenate was taken out as the whole cell fraction for subsequent protein immunoblot analysis, and the remaining homogenate was centrifuged twice at 1000g for 10 min and 5 min at 4°C to remove nuclei, cell debris and unbroken cells. Finally, the supernatant was collected and centrifuged at 10000g for 20 min at 4°C to obtain a mitochondrial-rich precipitate.

[0041] (2) Ultracentrifugation (UC): Cell culture, digestion, collection and disruption are the same as described in (1). The homogenate is centrifuged at 1000g for 10 min at 4°C and then centrifuged again for 5 min to remove cell nuclei, cell debris and broken cells. The supernatant is collected in a new centrifuge tube and centrifuged at 10,000g for 30 min to crudely extract mitochondria. The supernatant is discarded and the precipitate is resuspended in pre-cooled mitochondrial isolation reagent and placed on ice for subsequent ultracentrifugation. The specific process is as follows:

[0042] Add 15mL of 1.5M sucrose solution (1.5M sucrose, 10mM Tris-HCl, 1mM EDTA, pH 7.5) to an ultracentrifuge tube, tilt the ultracentrifuge tube at 45°, and vertically add 1M sucrose solution (1.0M sucrose, 10mMTris-HCl, 1mM EDTA, pH 7.5) to the upper layer of the 1.5M sucrose solution to form a 30mL sucrose gradient. Then, layer the mitochondrial suspension previously placed on ice on top of the 1M sucrose solution. Centrifuge at 8000g for 20min at 4°C. After the centrifugation is completed, carefully pipette the mitochondrial layer at the interface of the 1.5M sucrose solution and the 1M sucrose solution. Add diluent (5mM Tris-HCl, 1mM EDTA) to the collected mitochondrial solution, gradually increase the dilution, and dilute to 0.25M. The diluted mitochondrial suspension was centrifuged at 10,000 g for 20 min at 4°C to obtain a mitochondrial precipitate. The mitochondrial suspension was then gently washed once with pre-cooled KPBS and centrifuged at 10,000 g for 20 min at 4°C to obtain a mitochondrial precipitate.

[0043] 2. Sample Pretreatment

[0044] The sample pretreatment process was performed on ice. 400 μL of methanol extract containing internal standard was added to each mitochondrial sample extracted by differential centrifugation (DC) and ultracentrifugation (UC), and vortexed for 30 seconds. 1 mL of MTBE was added, vortexed for 30 seconds, and shaken at 1000 rpm and 10°C for 30 minutes. Then 400 μL of ultrapure water was added, vortexed for 1 minute, and centrifuged at 6°C and 12000g for 10 minutes. After the two phases were separated, the upper hydrophobic phase was quantitatively collected and vacuum dried in a refrigerated centrifugal concentrator. The resulting lyophilized samples were stored at -80°C. Before the lipid lyophilized samples were detected by ultra-performance liquid chromatography-mass spectrometry, they were re-dissolved with a methanol / dichloromethane mixed solution (v / v=1:2), and then diluted and injected with acetonitrile / isopropanol / water (v / v / v=65:30:5, containing 5 mM ammonium acetate).

[0045] 3. Data Collection

[0046] 1) Ultra-high performance liquid chromatography conditions: Thermo Fisher UltiMate 3000 (UPLC, Thermo, USA) system was used, and the chromatographic column was ACQUITY UPLC BEH C8 column (2.1×100mm×1.7μm, Waters, USA). During the separation process, the column temperature was set to 55°C, and the injection chamber temperature was controlled at 8°C. Mobile phase A was acetonitrile: ultrapure water = 6:4 (containing 10mM ammonium acetate), and mobile phase B was isopropanol: acetonitrile = 9:1 (containing 10mM ammonium acetate). The gradient elution mode was used, and the gradient was as follows: first, 32% B was maintained for 1.5min, then linearly increased to 85% B in the range of 1.5-15.5min, then linearly increased to 97% B within 0.1min and maintained for 18min, and then decreased to 32% B within 0.1min until equilibrium was reached for the next injection. The flow rate was maintained at 0.4mL / min throughout the process.

[0047] 2) Mass spectrometry conditions: A quadrupole and orbital trap hybrid Fourier transform ultra-high resolution mass spectrometer (Q-Exactive, Thermo, USA) was used, and the mass spectrometry analysis process was detected by electrospray ionization source in positive and negative ion modes. The mass spectrometry parameters were set as follows: spray voltage 3.0 kV, sheath gas flow rate 50 arb, auxiliary gas flow rate 15 arb, auxiliary gas temperature 400 ° C, capillary temperature 325 ° C, acquisition range 200-1800 m / z, acquisition rate 1.7 spectra / s, secondary analysis to collect the top 10 parent ions for HCD fragmentation mode, collision energy 25-35 eV. Xcalibur software (Thermo, USA) was used to record the total ion current and MS spectra during the acquisition process.

[0048] 4. Data Preprocessing

[0049] Importing the raw mass spectrometry data obtained by ULPC-Q Exative into MSDIAL

[0050] (http: / / prime.psc.riken.jp / compms / index.html) software was used for peak identification, peak extraction and peak alignment, and signal peaks with a signal-to-noise ratio lower than 10 were excluded. The resulting peak table was used to identify lipid substances using Xcalibular. The identified lipids met the following three conditions:

[0051] 1) Accurate mass number of primary mass spectrometry (theoretical value ± 5ppm);

[0052] 2) Secondary mass spectrometry MS / MS fragmentation rules;

[0053] 3) Chromatographic retention time rules.

[0054] The accurately identified lipid compounds were imported into TraceFinder (Thermo, USA) for compound quantification. Subsequently, the chromatographic peak intensity was corrected based on the total elution peak area of ​​the sample.

[0055] 5. Results Analysis

[0056] The structural formula and secondary mass spectra of lipid markers are as follows Figure 1 The correlation analysis of mitochondrial purity is shown in Figure 2 shown.

[0057] Using the statistical software SPSS, phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1; 2O (d18:1; 2O / 16:0), sphingomyelin SM 44:2; 2O (18:1; 2O / 24:1) and hexosylceramide HexCer42:2; 2O (d18:1; 2O / 24:1) were further calculated by binary logistic regression to obtain the joint marker P. The regression equation is as follows:

[0058] P=1 / (1+e^(-42.608a+5.583b-7.591c+7.343d+11.075))

[0059] Among them, a is the relative content of phosphatidylcholine PC 34:0 (16:0 / 18:0), b is the relative content of sphingomyelin SM34:1;2O (d18:1;2O / 16:0), c is the relative content of sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and d is the relative content of hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1). If P < 0.5, it is judged that the purity of mitochondria obtained by the purification method is high, and higher than the classical differential centrifugation method (DC), and comparable to the classical ultracentrifugation method (UC). If P > 0.5, it is judged that the purity of mitochondria obtained by the purification method is low, and lower than the classical ultracentrifugation method (UC), and comparable to the classical differential centrifugation method (DC).

[0060] All samples of the DC group and the UC group were included in the discriminant comparison, and the discriminant variable P( Figure 2 ), the area under the ROC curve (AUC value) for discrimination was 1, and at a cut-off value of 0.5, both the sensitivity and specificity were 100% (Table 1, Figure 2 c) The above results show that the marker has good discriminative potential and can be used to evaluate the purity of the extracted mitochondria. The P value obtained in the UC group is less than 0.5, and the P value obtained in the DC group is greater than 0.5, which proves that the purity of mitochondria obtained by ultracentrifugation (UC) is higher than that obtained by differential centrifugation (DC).

[0061] Western blot analysis of the DC and UC groups confirmed that the purity of mitochondria obtained by ultracentrifugation (UC) was higher than that obtained by differential centrifugation (DC) ( Figure 3 ).

[0062] Example 2

[0063] 1. Mitochondrial Sample Collection

[0064] (1) Column separation method (CO): The cell culture, digestion and collection process are the same as described in (1). Wash the collected cells twice with KPBS, add 250μL Buffer A (Invent, USA) and vortex to resuspend the cells. After vortexing, incubate on ice for 5 minutes. After incubation, vortex vigorously for 30 seconds, transfer the suspension into a centrifuge column, and centrifuge at 16000g for 30 seconds. Vortex to resuspend the precipitate, and centrifuge at 700g for 1 minute. Take the supernatant and transfer it to a new 2mL centrifuge tube. Add 300μL Buffer B (Invent, USA) and centrifuge at 16000g for 10 minutes. Discard the supernatant, add 200μL Buffer B (Invent, USA) to resuspend the precipitate, and centrifuge at 8000g for 5 minutes. Take the supernatant and transfer it to a new 2mL centrifuge tube, add 1600μL pre-cooled KPBS, and centrifuge at 16000g for 30 minutes to obtain a mitochondrial-rich precipitate.

[0065] (2) Magnetic bead method (MACS): The cell culture, digestion and collection procedures were the same as described in (1). The cells were washed three times with pre-cooled KPBS. TM Anti-HA magnetic beads (Thermo Fisher Scientific, USA) were placed on ice for later use. The collected cells were washed twice with pre-cooled KPBS, and 1 mL of KBPS was added to resuspend the cell pellet. Transfer to a Dounce homogenizer and grind 45 times using a tight hammer. Then the homogenate was transferred to a new 2 mL centrifuge tube and centrifuged at 2000 g for 5 min at 4 °C to remove cell nuclei, cell debris, and broken cells. The supernatant was collected and placed in a pre-washed Pierce TM Place the Anti-HA magnetic beads in a centrifuge tube. Mix by gently pipetting and incubate on a shaker at 4°C for 3.5 minutes. Spin the beads to allow the solution hanging on the tube wall to enter the tube. Place the tube on a magnetic rack and discard the liquid after the solution is clear. Wash the beads gently 4 times with pre-cooled KPBS and finally place them in Pierce TM Anti-HA magnetic beads contain mitochondria.

[0066] 2. Sample Pretreatment

[0067] The sample pretreatment process was performed on ice. 400 μL of methanol extract containing internal standard was added to each mitochondrial sample obtained by column separation (CO) and magnetic bead method (MACS), and vortexed for 30 seconds. 1 mL of MTBE was added, vortexed for 30 seconds, and shaken at 1000 rpm and 10°C for 30 minutes. Then 400 μL of ultrapure water was added, vortexed for 1 minute, and centrifuged at 6°C and 12000g for 10 minutes. After the two phases were separated, the upper hydrophobic phase was quantitatively collected and vacuum dried in a refrigerated centrifugal concentrator. The resulting freeze-dried samples were stored at -80°C.

[0068] 3. Data collection: same as Example 1.

[0069] 4. Data preprocessing: same as Example 1.

[0070] 5. Results Analysis

[0071] Based on the comparison between the CO group and the MACS group, Example 2 was used to verify the feasibility of the combined use of the above two lipid markers to further confirm their discriminative effect.

[0072] All samples of CO group and MACS group were included in the discriminant comparison, and the discriminant variable P( Figure 2 ), the area under the ROC curve (AUC value) for discrimination was 1, and at a cut-off value of 0.5, both the sensitivity and specificity were 100% (Table 1, Figure 2 d), the above results show that the marker has good discriminative potential and can be used to discriminate the purity of the extracted mitochondrial samples. Among them, the P value of the MACS group is less than 0.5, and the P value of the CO group is greater than 0.5, which proves that the purity of the mitochondria extracted by the MACS group is higher than that of the mitochondria extracted by the CO group, and the column separation method (CO) is comparable to the classical differential centrifugation method (DC), and the magnetic bead method (MACS) is comparable to the classical ultracentrifugation method (UC).

[0073] Western blot analysis of the CO and MACS groups confirmed that the purity of mitochondria extracted by the magnetic bead method (MACS) was higher than that extracted by the column separation method (CO), and the results of the column separation method (CO) were comparable to those of the classical differential centrifugation method (DC), and the results of the magnetic bead method (MACS) were comparable to those of the classical ultracentrifugation method (UC). Figure 3 ).

[0074] Table 1: ROC discriminant analysis results of markers

[0075]

Claims

1. A combined metabolic marker for evaluating mitochondrial purity, characterized in that The combined metabolic markers include phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1; 2O (d18:1; 2O / 16:0), sphingomyelin SM 44:2; 2O (18:1; 2O / 24:1) and hexosylceramide HexCer42:2; 2O (d18:1; 2O / 24:1).

2. Use of the combined metabolic marker according to claim 1 in the evaluation of mitochondrial purity obtained by a mitochondrial extraction method, wherein the extraction method comprises differential centrifugation, column separation, ultracentrifugation and magnetic bead method.

3. The method for determining the purity of mitochondria extracted by the mitochondrial extraction method using the combined metabolic markers according to claim 1, characterized in that: This method uses the combined marker variable P to make judgments: P=1 / (1+e^(-42.608a+5.583b-7.591c+7.343d+11.075)) Among them, a is the relative content of phosphatidylcholine PC 34:0 (16:0 / 18:0), b is the relative content of sphingomyelin SM34:1;2O (d18:1;2O / 16:0), c is the relative content of sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and d is the relative content of hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1). If P < 0.5, it is judged that the purity of mitochondria proposed by this method is higher than that of the classical differential centrifugation method, and is comparable to the classical ultracentrifugation method; if P > 0.5, it is judged that the purity of mitochondria proposed by this method is lower than that of the classical ultracentrifugation method, and is comparable to the classical differential centrifugation method.

4. A kit for determining the purity of mitochondria extracted by a mitochondrial extraction method, characterized in that The kit comprises: Standards: ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0), and sphingomyelin SM 30:1;2 (d18:1; / 12:0); Pretreatment extract: methanol solution containing internal standards of 1.27 μg / mL ceramide Cer 35:1;2 (d18:1 / 17:0), 2.17 μg / mL phosphatidylcholine PC 38:0 (19:0 / 19:0) and 1.38 μg / mL sphingomyelin SM 30:1;2 (d18:1; / 12:0); in positive ion detection mode, ceramide Cer 35:1;2 (d18:1 / 17:0) is used to calibrate ceramide and hexosylceramide, sphingomyelin SM 30:1;2 (d18:1; / 12:0) is used to calibrate sphingomyelin, and phosphatidylcholine PC38:0 (19:0 / 19:0) is used to calibrate phosphatidylcholine in negative ion detection mode; Eluent: Mobile phase A is an acetonitrile / water solution containing 10 mM ammonium acetate (v / v=6:4), and mobile phase B is an isopropanol / acetonitrile solution containing 10 mM ammonium acetate (v / v=9:1).

5. A method for calculating a combined marker variable using the kit according to claim 4, characterized in that The following steps are involved: 1) Treat the mitochondrial samples obtained by each method with an extraction solution containing an internal standard, precipitate the protein, and then extract the lipid metabolites; 2) separating and identifying lipid metabolites of the test sample extracted after the treatment in step 1) based on ultra-high performance liquid chromatography-mass spectrometry; 3) Use the ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0) and sphingomyelin SM 30:1;2 (d18:1; / 12:0) standards in the kit to assist in qualitative confirmation of the detected ions; 4) The chromatographic peak intensities of the four substances identified in each test sample were corrected by the total mitochondrial sample outflow peak area to obtain the relative concentrations of the four substances; 5) The relative concentration values ​​of the above two metabolites were used to calculate the combined marker variable P based on the binary logistic regression equation.

6. The method for calculating the combined marker variable using the kit according to claim 5, characterized in that: The method uses ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0) and sphingomyelin SM 30:1;2 (d18:1; / 12:0) standards to assist in qualitative confirmation of the detected ions, and specifically includes the following steps: 1) The ceramide Cer 35:1; 2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0) and sphingomyelin SM 30:1; 2 (d18:1; / 12:0) standards in the kit were analyzed by ultra-performance liquid chromatography-mass spectrometry to determine the chromatographic retention time, measured mass-to-charge ratio, and secondary mass spectrometry characteristic ions of the standards. Combined with the Lipid Maps database and the ceramide Cer 35:1; 2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0) and sphingomyelin SM 30:1;2 (d18:1; / 12:0) standard, determine the secondary mass spectrometry bond breaking rules of ceramide, phosphatidylcholine and sphingomyelin: in positive ion mode, the characteristic secondary of ceramide is LCB chain, which is determined according to its dehydrated number and chain structure; sphingomyelin undergoes neutral loss, and a characteristic ion with m / z of 184 can be obtained; in negative ion mode, the acyl chain of phosphatidylcholine breaks, generating characteristic fatty acid ions corresponding to different sites, and the corresponding characteristic ions are found in the secondary mass spectrometry according to the m / z of the fatty acid ions; 2) In the tested mitochondrial samples, hexosylceramide HexCer42:2;2O The extraction parameters of (d18:1; 2O / 24:1) were: positive ion mode, mass-to-charge ratio of 782.65039±5ppm; the extraction parameters of sphingomyelin SM34:1;2O (d18:1; 2O / 16:0) and sphingomyelin SM 44:2;2O (18:1; 2O / 24:1) were: mass-to-charge ratio of 703.57489±5ppm and 841.7157±5ppm respectively; the extraction parameters of phosphatidylcholine PC 34:0 (16:0 / 18:0) were: negative ion mode, mass-to-charge ratio of 820.6073±5ppm; 3) Check whether the secondary mass spectrometry ions of the above chromatographic peaks conform to the characteristic secondary mass spectrometry bond breaking rules, and confirm the target lipids in combination with the chromatographic retention behavior.

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