A method for precise metabolic flux analysis of TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry

Abstract

The application belongs to the field of biological analysis, and provides a method for precise metabolic flow analysis of TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry. By optimizing mass spectrometry parameters and liquid chromatography method, one injection within 6 min is realized, and high-throughput, high-sensitivity and high-resolution detection is realized on 11 biomarkers related to TCA cycle metabolic pathways. At the same time, the application innovatively uses LC-MS / MS to decipher 13 C the stepwise position-specific transfer from glucose to subsequent metabolites by glycolysis and TCA cycle, thereby calculating 13 C the integrated steady-state analysis of key metabolic rates of position-specific transfer from sequential precursors to their products (pyruvate dehydrogenase, beta-oxidation, pyruvate carboxylase, isocitrate dehydrogenase and pyruvate cycle). This technology has wide applicability and can potentially characterize mitochondrial metabolism of any tissue or cell.

Description

Technical Field

[0001] This invention belongs to the field of bioanalysis and relates to a method for precise metabolic flux analysis of the TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry. Background Technology

[0002] Since the discovery of DNA, human understanding of genes and their protein products has continuously increased, and research in the biological field has been constantly advancing. In the past decade, with the continued prevalence of obesity and metabolic syndrome, interest in metabolomics has grown, and metabolism has gradually taken a mainstream position in many areas of biology. Today, more and more research is analyzing from a metabolic perspective. Overall, with the continuous advancement of metabolite measurement technologies (especially mass spectrometry), metabolomics has profound research value.

[0003] From bacteria to humans, the core of oxidative and anabolistic metabolism revolves around the continuous enzymatic reactions of the citric acid cycle (TCA cycle), which forms the cornerstone of the body's metabolism. Mitochondria contain numerous interwoven reaction nodes, and the exchange, replenishment, and dispersion of metabolites at these nodes complicate metabolic flux analysis. Metabolic activity can be quantified by the flow rate of substances per unit time, i.e., metabolic flux. However, measuring metabolite concentration is not equivalent to metabolite flux. Current research methods cannot accurately analyze the flux of metabolites in complex metabolic reactions. This may be because, during metabolism, the accumulation of metabolites is not only due to increased production but may also be due to decreased consumption. For example, when glucose is removed from yeast, although the inflow to the glycolysis pathway decreases, the outflow increases sharply, reducing the glycolysis flux. In this case, the increase in metabolite concentration in the outflow does not match the decrease in glycolysis flux. Since metabolite levels and fluxes provide more comprehensive information, researchers best achieve a comprehensive understanding of metabolism by integrating both analyses.

[0004] Mass spectrometry offers the potential to simultaneously measure labeled and unlabeled metabolites, improve sensitivity, and analyze single isotopic isomers. Here, we propose a comprehensive and direct metabolic flux platform that uses a precise metabolic flux method based on isotope tracing combined with high-resolution mass spectrometry to track and analyze the stepwise, position-specific transfer of mass isotope labels in the continuous reactions of glycolysis and the TCA cycle. This technique primarily involves direct deconvolution from… 13 Mass spectrometry information of citrate produced from C-labeled glucose, lactate, and other glucogenic substrates can be used to decipher the position-specific isotopic enrichment of TCA cycle metabolic intermediates such as acetyl-CoA, oxaloacetate, and citrate in mitochondria. Simultaneously, it can calculate... 13Key metabolic processes involved in the position-specific transfer of glucose from its precursors to its products (such as pyruvate dehydrogenase, β-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and the pyruvate cycle) are analyzed to assess overall cellular homeostasis. Notably, the deconvolution technique using mass spectrometry information of intermediate metabolites in the TCA cycle, such as citrate, overcomes the significant limitation of previous isotope analysis techniques, which could only determine the concentration of the target analyte but not its specific flux. Furthermore, this technique has broad applicability to any glucose-oxidizing cell, providing a new analytical method for analyzing the mechanisms by which analyte conditions affect glucose metabolism and for exploring unknown metabolic pathways. Summary of the Invention

[0005] The technical problem to be solved: Quantitative calculation of metabolites in cellular mitochondria requires measuring the flux between metabolic reactions. Due to the numerous intersecting reaction nodes, the exchange, replenishment, and dispersion of metabolites at these nodes complicate isotope labeling. However, current analytical methods cannot accurately detect the isotope flow in these complex metabolic reactions. The inventors used LC-MS / MS to stepwise analyze the isotopes from glucose to subsequent metabolites in glycolysis and the TCA cycle. 13 C) Position-specific transfer: By deciphering citrate isotope isomers to unlock cross-functional metabolic fluxes, this allows for comprehensive localization of precursor-to-product metabolic reactions. 13 Changes in C-tag transfer allow for precise quantification of the exchange rates between oxidation, complementation, cyclic reactions, and metabolites in mitochondria.

[0006] Technical Solution: This invention provides a method for precise metabolic flux analysis of the TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry, comprising the following steps:

[0007] (1) Preparation of culture medium:

[0008] The method for preparing sugar-free culture medium is as follows: add 0.1-2.0% BSA, 12-30mM sodium bicarbonate, and 5-15mM HEPES to DMEM medium (sigma-D5030), mix well with a magnetic stir bar, and adjust the pH to 6.8-7.8.

[0009] The high-glucose medium (first medium) is prepared as follows: 0.1–2.0% BSA, 12–30 mM sodium bicarbonate, 5–15 mM HEPES, and 1–25 mM glycogen substrate (glucose substrate is glucose, lactic acid, pyruvate, or glutamine) are added to DMEM medium (sigma-D5030). The mixture is stirred with a magnetic stir bar and the pH is adjusted to 6.8–7.8. If the cells are hepatocytes, the glycogen substrate is 1–20 mM lactic acid and 1–20 mM pyruvate; if the cells are pancreatic islet cells, the glycogen substrate is 1–20 mM glucose and 1–20 mM glutamine.

[0010] The prepared culture medium was filtered through a 0.22 μM filter membrane, and 0.1% penicillin-streptomycin mixture was added. It was then stored at 4°C.

[0011] 13 The preparation method of C-labeled high-glucose medium (second medium) is as follows: Add 0.1–2.0% BSA, 12–30 mM sodium bicarbonate, 5–15 mM HEPES, and 1–25 mM sodium bicarbonate to DMEM medium (Sigma-D5030). 13 C-labeled glycogenic substrate (glycogenic substrate is [U- 13 C6] glucose, [4,5- 13 C2] glutamine or [U- 13 [C3] Lactic acid, [U- 13 [C3] Pyruvic acid), mix well with a magnetic stir bar, and adjust the pH to 6.8-7.8;

[0012] The prepared culture medium was filtered through a 0.22 μM filter membrane and 0.1% penicillin-streptomycin mixture was added. It was then stored at 4 °C.

[0013] (2) Cell culture:

[0014] When the cell density reached 80%, the cells were digested and cultured in complete medium (80% high glucose medium + 10% FBS) at a concentration of 5 × 10⁻⁶. 5 Seeds were placed into six-well plates at a density of 10 cells / well. Once the cells adhered and reached 80% density, they were starved in sugar-free medium for 1–4 hours, then cultured in the first medium for 1–6 hours to achieve metabolic homeostasis. Finally, cells were treated with isotope-containing medium. 13 The second culture medium labeled with C reached cell isotope homeostasis after 1–6 hours.

[0015] *The timing of marking the latency period varies from cell to cell. After conducting preliminary studies on the quenching time process, the point at which the cell reaches isotopic homeostasis can be determined.

[0016] (3) Cell quenching:

[0017] Pre-chill 5 mM HEPES, quenching buffer (10–40% methanol, 0.1–1% formic acid, 0.5–5 mM sodium fluoride, 1–2 mM phenylalanine, and 50–150 μM ethylenediaminetetraacetic acid) and V-type 96-well plates on ice. After cell culture, place the 96-well plates on ice, quickly aspirate the culture medium, add 2 mL of pre-chilled (4°C) 5 mM HEPES to each well to wash the cells, then aspirate them, add 150 μL of ice-cold quenching buffer, scrape the cells off the culture medium with a spatula, and quickly transfer the cell and quenching buffer mixture to the ice-cold 96-well plates. Seal the 96-well plates with aluminum foil, poke a hole in each well with a 20 g needle, store them in a -80°C freezer overnight, and then freeze-dry them.

[0018] (4) Preparation of liquid chromatography-mass spectrometry (LC-MS) samples:

[0019] Before performing mass spectrometry analysis, prepare the sample by pre-cooling the centrifuge to 4°C, placing the V-type 96-well plate on ice, resuspending the lyophilized cell powder in pre-cooled 50 μL of ultrapure water (containing 2–100 μM taurine), and centrifuging the 96-well plate at 4000 rpm for 5 min. Transfer the supernatant to a new 96-well plate and repeat the above operation at least 3 times to ensure that there is no precipitate in the supernatant. Transfer it to a sample vial. For quality control, create a mixed sample well by taking 2–3 μL from each well.

[0020] (5) Liquid chromatography-mass spectrometry analysis

[0021] A single standard was dissolved in 50% 1M HCl and 50% methanol to prepare a 5mM stock solution. On the day of the experiment, a mixed standard was prepared by serially diluting the mixed standard with ultrapure water containing 25μM taurine (internal standard) to 200 / 100 / 50 / 25 / 12.5 / 6.75 / 3.125 / 1.5625 / 0.78125μM to plot a standard curve. After the analysis of 10 samples, the mixed sample well was analyzed once. Mass spectrometry in multiple reaction monitoring mode (MRM mode) was used to monitor the stable isotopes. 13 Mass spectrometry analysis was performed on the cell sample extract cultured in C-labeled medium. Liquid chromatography-mass spectrometry analysis of the target metabolites was performed in a Waters Acquity UPLC system under the following chromatographic conditions:

[0022] Chromatographic column: ACQUITY UPLC@HSS T3 column, 2.1mm×100mm, 1.8μm;

[0023] Mobile phases: Phase A: 0.1% formic acid aqueous solution, and Phase B: 0.1% formic acid acetonitrile solution;

[0024] The liquid phase gradient program was: 0.0–0.5 min, 99% A; 0.5–4.0 min, 99%–95% A; 4.01–6.0 min, 99% A.

[0025] Column temperature: 20~55℃;

[0026] Sample cell temperature: 4~10℃;

[0027] Mobile phase flow rate: 0.2–0.5 mL / min;

[0028] Injection volume: 1 μL;

[0029] Mass spectrometry detection was performed using a Xevo TQ-S mass spectrometer in electrospray ionization (ESI) negative ion detection mode. The source parameters for the mass spectrometry analysis method are as follows:

[0030] Capillary voltage in negative ion mode: 0.2~3.2kV;

[0031] The cone voltage and collision voltage settings depend on the specific MRM channel for each metabolite (as shown in Table 1 below);

[0032] Desolventizing gas flow rate: 800~1200L / h;

[0033] Temperature: 450~600℃;

[0034] Conical orifice gas flow rate: 100~250L / h;

[0035] Atomizer setting: 5-10 Bar;

[0036] In MRM mode, parent / daughter ions are screened simultaneously to collect data. All of the above operations are controlled by MassLynx software.

[0037] Table 1

[0038]

[0039]

[0040]

[0041] (6) Background correction of liquid chromatography-mass data

[0042] Export peak area data from MassLynx software, using each 13 The peak area of ​​the C-labeled sample minus the ordinary 12 The peak area of ​​sample C is the peak area of ​​each sample after background correction.

[0043] (7) Natural abundance correction

[0044] In order to remove from nature 13 The influence of C requires the measured values ​​to be... 13 C was used to correct for the natural abundance to obtain the experimentally added... 13 C abundance, natural abundance adjusted by 1.1%, corrected isotope matrix I' (Pm，Dn) This is to explain the presence of naturally abundant carbon in each possible combination of mother / daughter ions in the matrix. (Pm，Dn) :

[0045] I′(P m D n )=I(P m D n )*(1+K(pm))-I(P m-1 D n )*k((pd)-(mn-1 )) -I(P m-1 D n-1 )*k(d-(n-1))

[0046] p: Total number of carbons in the parent ion;

[0047] d: The total number of carbons in the daughter ions;

[0048] m: in the parent ion 13 The number of C;

[0049] n: in the daughter ion 13 The number of C;

[0050] I: Peak area corresponding to the parent ion P from 0→p and the daughter ion D from 0→d;

[0051] k = 0.11 (in nature) 13 (Natural abundance of C);

[0052] mv≤pd.;

[0053] (8) Deconvolution of citric acid

[0054] 13 C glucose will produce 13 C-pyruvate enters the TCA cycle via the pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PC) pathways. Citrate (Cit) is a symmetrical molecule, but it contains a prochiral center and can be distinguished by the stereochemical characteristics of tricarboxylic acid cycle enzymes (e.g., Figure 2 (As shown); Acetyl-CoA (AcCOA) provides C4 and C5 of citrate, and oxaloacetate (OAA) provides C1, 2, 3, and 6. If all the C4 and C5 are available, then... 13 C-labeled Cit family (Cit) a,d,h,f,i,j ,like Figure 3As shown in the diagram, carbon atoms generated by the PDH pathway can be resolved. Similarly, carbon atoms generated by the PC pathway can also be resolved by... 13 C-labeled Cit family (Cit) c,h )express;

[0055] In the fragmentation mode, removing C1, C6 or C5, C6 of Cit yields daughter ions, because these two carbon atoms contain information from the PC and PDH pathways. Deconvolving all daughter ions that have lost two carbon atoms yields metabolic flux information for the Cit family.

[0056] Without 13 The C-marked Cit is M, with 1 to 6 _ 13 The C-labeled Cit values ​​are represented as [M+2], [M+3], [M+4], [M+5], and [M+6], respectively. The parent / daughter combination assignments for individual [M+2] to [M+6] isotope groups are as follows:

[0057] There are two ways to generate Cit[M+2] with two tags: one is from the PDH pathway. a Secondly, the Cit generated from the PDH pathway in the second TCA cycle. b Cit a Fragments of the group can produce equal amounts of 193 / 68 and 193 / 69 daughter ions, Cit b Fragments of the group can produce 3 / 4 of the 193 / 68 daughter ions and 1 / 4 of the 193 / 69 daughter ions, from which we can derive the equation describing the isotopic composition of the parent / daughter combination:

[0058]

[0059]

[0060] Equations 1 and 2 can be solved to find:

[0061] Cit a = 3 * 193 / 69 - 193 / 68 (Equation 3)

[0062] Cit b = 2*(193 / 68-193 / 69) (Equation 4)

[0063] Similarly, we can conclude that:

[0064] Cit c = 3*194 / 69 - 5*194 / 68 - 5*194 / 70 (Equation 5)

[0065] Cit d= 4*194 / 68 - 4*194 / 69 + 12*194 / 70 (Equation 6)

[0066] Cit e = 2*194 / 68 - 2*194 / 69 - 6*194 / 70 (Equation 7)

[0067] Cit f = 2*195 / 70 + 2*195 / 69 (Equation 8)

[0068] Cit g = 9 * 195 / 69 - 195 / 70 (Equation 9)

[0069]

[0070]

[0071] Cit j =197 / 71 (Equation 12);

[0072] (9) Correction of isocitrate dehydrogenase

[0073] The deconvolution process assumes a direct flow of carbon from Cit to α-ketoglutarate (αKG). However, due to the presence of isocitrate dehydrogenase (ICDH), this reaction is reversible, and αKG can also be converted back to Cit. This reverse flux affects the isotopic labeling pattern of Cit. This ICDH reverse flux only affects C6-containing... 13 If the C-labeled Cit is unaffected, while the C4 and C5 labels are unaffected, then the correlation of this flux is with all [4,5- 13 C]Cit family (∑Cit) a Cit f Cit i Cit h Cit d Cit j ) and [1,2- 13 The sum of C2]AcCOA is directly proportional, defined as Φ AcCit (Isotope steady-state relationship):

[0074]

[0075] The following equations use ΦAcCit to correct the Q1 / Q3 segments of each Cit to eliminate the reverse ICDH flux and remove the effects of the reverse flux:

[0076]

[0077]

[0078]

[0079] 195 / 70 c =195 / 70*196 / 70(1-Φ AcCit )+195 / 69(1-Φ AcCit (Equation 17)

[0080]

[0081] 194 / 70 c =194 / 70-195 / 69(1-Φ) AcCit (Equation 19)

[0082] 194 / 69 c =194 / 69-195 / 69(1-Φ) AcCit )+194 / 68(1-Φ AcCit (Equation 20)

[0083]

[0084] 193 / 69 c =193 / 69-194 / 68(1-Φ) AcCit (Equation 22)

[0085] 193 / 68 c =193 / 68-194 / 68(1-Φ) AcCit )+(193 / 68-193 / 69)(1-Φ AcCit (Equation 23)

[0086] (10) Mass Isotope Analysis Distribution - Calculation of Isotopic Enrichment of Acetyl-CoA and Oxaloacetate

[0087] If pyruvate and AcCOA enrichment is known, the relative contributions of glucose oxidation and β-oxidation to AcCOA used by citrate synthase (CS) can be determined. In formal experiments, it may be difficult for researchers to directly measure the fractional enrichment of AcCOA and OAA in the mitochondrial matrix pool. This invention can solve this problem through mass isotope distribution analysis (MIDA).

[0088] For the reaction A + B → AB, if both substrates are partially enriched (FE) A* and FE B* ), then MIDA will determine the enrichment of the two precursors, even in the presence of dilution from externally unlabeled products, the partial enrichment (FE) of the matrix is ​​defined as:

[0089]

[0090]

[0091] (*) indicates the presence of a measurable label.

[0092] (A+A * ) and (B+B * The reaction will produce AB + AB. * +A * B+A * B * =1.

[0093] If unlabeled contaminant A'B' exists, then AB + AB * +A * B+A * B * +A'B' = 1.

[0094] Generate dual-labeled products (D A*B* ) and single-labeled substrate (S A*B and S AB* The probability of forming a product is determined by the fractional enrichment of the product:

[0095]

[0096]

[0097]

[0098] Equations 29 and 30 below describe the ratio of single-labeled products to dual-labeled products:

[0099]

[0100]

[0101] Solving the above equation yields the equation for FE:

[0102]

[0103]

[0104] Cit is formed in the mitochondrial matrix by condensing AcCOA and OAA using CS. Mass spectrometry can evaluate individual molecules, so D can be determined from the deconvolution of isotope families. A*B* S A*B and S AB* (See deconvolution of citrate isotopes), the inventors discovered several possible methods to calculate labeled AcCOA (FE) in the mitochondrial matrix. A*) and marked OAA (FE) B* The partial enrichment of OAA and AcCOA. In summary, the steady-state enrichment of OAA and AcCOA can be calculated by analyzing the enrichment of several citrate isotope isomers, since any given citrate isotope isomer is a result of the enrichment of OAA and AcCOA, for example, [U- 13 C6]Cit is [U- 13 C4]OAA and [1,2- 13 The product of C2]AcCOA;

[0105] [1,2- 13 The calculation method for C2]AcCOA is as follows:

[0106]

[0107] 13 The equation for calculating OAA with C-label is as follows:

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] (11) Calculation of metabolic flux ratio based on isotopic steady state

[0114] Steady-state isotope analysis can determine whether there is a significant net inflow or exchange of unlabeled metabolites between successive or tandem metabolic reactions. One or more metabolic pathways may involve enzymatic reactions that produce products; for example, AcCOA formation primarily results from PDH or the β-oxidation of fatty acids or certain amino acids. If one pathway can be selectively labeled (e.g., pyruvate dehydrogenation or pyruvate carboxylation), the relative source of carbon inflow can be determined. Differential equations can be used to describe the rate of change in substrate-labeled metabolite enrichment due to metabolic inflow (inflow rate minus efflow rate). For generalized reactions:

[0115]

[0116] Where A is the initial substrate converted to product B by enzyme E1, and then B is converted to C by enzyme E2, the general equation is:

[0117]

[0118] Under metabolic and isotopic stability conditions 13The change in C concentration over time is defined as zero. Therefore, the pathway from E1 to E2 with respect to (Φ 1→2 The solution can be obtained in such a way that the relative contribution of the input to the output is equal to the enrichment of the product on its precursor.

[0119]

[0120] If V E1 If V is the only path that helps generate B, then V E1 / V E2 The value will approach 1. However, this steady-state isotope analysis can only determine whether there is a significant net entry or exchange of unlabeled metabolites between sequential or tandem metabolic reactions (entry and exit are in equilibrium), and a value less than 1 indicates unlabeled input from another source. In simpler terms, Φ AB This refers to the relative contribution of substrate A to the pathway leading to product B, i.e., the metabolic rate ratio, while 1-Φ AB This indicates an unlabeled input to that pathway. It's important to note that this analysis cannot distinguish between reaction-replacement and exchange reactions, nor can it identify carbon losses from reaction-replacement. Attached Figure Description

[0121] Figure 1 The experimental procedure of this invention involves isotope labeling of cells followed by liquid chromatography-mass spectrometry (LC-MS) analysis, and finally, the data analysis yields the precise metabolic flow of the isotopes.

[0122] Figure 2 The serial numbers of the carbon atoms in citric acid are used to name the carbon atoms in this invention.

[0123] Figure 3 Characterization of the parent ion and daughter ion fragments of the citric acid deconvolution isotope isomers in this invention;

[0124] Figure 4 This is a schematic diagram of the metabolic pathway centered on acetyl-CoA in Example 2 of the present invention, in which β-oxidation and pyruvate dehydrogenase pathways flow into acetyl-CoA, while citrate synthase pathway flows out of acetyl-CoA.

[0125] Figure 5 In Example 2 of this invention, glucose oxidation (left figure) and V were used respectively. PDH / V CS (Right figure) shows the results of measuring the muscle metabolic sensitivity of rats under insulin resistance.

[0126] Figure 6 This is a schematic diagram of the metabolic pathway centered on oxaloacetate / malic acid in Example 3 of the present invention, in which pyruvate decarboxylase and citrate synthase pathways both flow into oxaloacetate / malic acid, while phosphoenolpyruvate carboxylkinase and citrate synthase pathways flow out of oxaloacetate / malic acid.

[0127] Figure 7 The changes in INS-1 insulin secretion (A) and Φ under different glucose concentrations in Example 3 of this invention. PO Change (B) diagram. Detailed Implementation

[0128] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0129] Example 1

[0130] A method for precise metabolic flux analysis of TCA circulation in pancreatic islet cells based on isotope tracing technology combined with high-resolution mass spectrometry includes the following steps:

[0131] (1) Preparation of culture medium:

[0132] The method for preparing sugar-free culture medium is as follows: add 0.2% BSA, 24mM sodium bicarbonate, and 10mM HEPES to DMEM medium (sigma-D5030), mix well with a magnetic stir bar, and adjust the pH to 7.4.

[0133] The high-glucose medium (first medium) was prepared as follows: 0.2% BSA, 24mM sodium bicarbonate, 10mM HEPES and 20mM glucose were added to DMEM medium (sigma-D5030), mixed with a magnetic stir bar, and the pH was adjusted to 7.4.

[0134] The prepared culture medium was filtered through a 0.22 μM filter membrane, and 0.1% penicillin-streptomycin mixture was added. It was then stored at 4°C.

[0135] 13 The method for preparing C-labeled high-glucose medium (second medium) is as follows: Add 0.2% BSA, 24mM sodium bicarbonate, 10mM HEPES, and 20mM sodium bicarbonate to DMEM medium (sigma-D5030). 13 C-labeled glucose was mixed with a magnetic stir bar and the pH was adjusted to 7.4.

[0136] The prepared culture medium was filtered through a 0.22 μM filter membrane and 0.1% penicillin-streptomycin mixture was added. It was then stored at 4°C.

[0137] (2) Pancreatic islet cell culture:

[0138] When the islet cell density reached 80%, the islet cells were digested and cultured in complete medium (80% high glucose medium + 10% FBS) at a concentration of 5 × 10⁻⁶. 5Seeds were placed at a density of 10 cells / well into six-well plates. Once the islet cells adhered and reached a density of 80%, they were starved in sugar-free medium for 2 hours, then cultured in the first medium for 3 hours to achieve metabolic homeostasis. Finally, islet cells were treated with isotope-containing medium. 13 The second culture medium labeled with C for 3 hours reached cell isotope homeostasis.

[0139] (3) Cell quenching:

[0140] Pre-chill 5 mM HEPES, quenching buffer (20% methanol, 0.1% formic acid, 3 mM sodium fluoride, 1 mM phenylalanine, and 100 μM ethylenediaminetetraacetic acid) and V-type 96-well plates on ice. After islet cell culture, place the 96-well plates on ice, quickly aspirate the culture medium, add 2 mL of pre-chilled (4°C) 5 mM HEPES to each well to wash the islet cells, then aspirate them, add 150 μL of ice-cold quenching buffer, scrape the islet cells off the culture medium with a spatula, and quickly transfer the islet cell and quenching buffer mixture to the ice-cold 96-well plates. Seal the 96-well plates with aluminum foil, poke a hole in each well with a 20 g needle, store them in a -80°C freezer overnight, and then freeze-dry them.

[0141] (4) Preparation of liquid chromatography-mass spectrometry (LC-MS) samples:

[0142] Samples were prepared before mass spectrometry analysis. The centrifuge was pre-cooled to 4°C, and a V-type 96-well plate was placed on ice. The lyophilized islet cell powder was resuspended in pre-cooled 50 μL of ultrapure water (containing 25 μM taurine), and the plate was centrifuged at 4000 rpm for 5 min. The supernatant was transferred to a new 96-well plate, and the above operation was repeated at least 3 times to ensure that there was no precipitate in the supernatant. The supernatant was then transferred to a sample vial. For quality control, a mixed sample well was created by drawing 2.50 μL from each well.

[0143] (5) Liquid chromatography-mass spectrometry analysis

[0144] A single standard was dissolved in 50% 1M HCl and 50% methanol to prepare a 5mM stock solution. On the day of the experiment, a mixed standard was prepared by serially diluting the mixed standard with ultrapure water containing 25μM taurine (internal standard) to 200 / 100 / 50 / 25 / 12.5 / 6.75 / 3.125 / 1.5625 / 0.78125μM to plot a standard curve. After the analysis of 10 samples, the mixed sample well was analyzed once. Mass spectrometry in multiple reaction monitoring mode (MRM mode) was used to monitor the stable isotopes. 13 Mass spectrometry analysis was performed on the cell sample extract cultured in C-labeled medium. Liquid chromatography-mass spectrometry analysis of the target metabolites was performed in a Waters Acquity UPLC system under the following chromatographic conditions:

[0145] Chromatographic column: ACQUITY UPLC@HSS T3 column, 2.1mm×100mm, 1.8μm;

[0146] Mobile phases: Phase A: 0.1% formic acid aqueous solution, and Phase B: 0.1% formic acid acetonitrile solution;

[0147] The liquid phase gradient program was: 0.0–0.5 min, 99% A; 0.5–4.0 min, 99%–95% A; 4.01–6.0 min, 99% A.

[0148] Column temperature: 35℃;

[0149] Sample cell temperature: 4℃;

[0150] Mobile phase flow rate: 0.4 mL / min;

[0151] Injection volume: 1 μL;

[0152] Mass spectrometry detection was performed using a Xevo TQ-S mass spectrometer in electrospray ionization (ESI) negative ion detection mode. The source parameters for the mass spectrometry analysis method are as follows:

[0153] Capillary voltage in negative ion mode: 2.50kV;

[0154] The cone voltage and collision voltage settings depend on the specific MRM channel for each metabolite (as shown in Table 1 below);

[0155] Desolventizing gas flow rate: 1000 L / h;

[0156] Temperature: 550℃;

[0157] Conical orifice gas flow rate: 150 L / h;

[0158] Atomizer setting: 7.0 Bar;

[0159] In MRM mode, data is collected by simultaneously screening parent / daughter ions. All of the above operations are controlled by MassLynx software.

[0160] Table 1

[0161]

[0162]

[0163] (6) Background correction of liquid chromatography-mass data

[0164] Export peak area data from MassLynx software, using each 13 The peak area of ​​the C-labeled sample minus the ordinary 12The peak area of ​​sample C is the peak area of ​​each sample after background correction.

[0165] (7) Natural abundance correction

[0166] In order to remove from nature 13 The influence of C requires the measured values ​​to be... 13 C was used to correct for the natural abundance to obtain the experimentally added... 13 C abundance, natural abundance adjusted by 1.1%, corrected isotope matrix I' (Pm，Dn) This is to explain the presence of naturally abundant carbon in each possible combination of mother / daughter ions in the matrix. (Pm，Dn) :

[0167] I′(P m D n )=I(P m D n )*(1+K(pm))-I(P m-1 D n )*k((pd)-(mn-1))-I(P m-1 D n-1 )*k(d-(n-1))

[0168] p: Total number of carbons in the parent ion;

[0169] d: The total number of carbons in the daughter ions;

[0170] m: in the parent ion 13 The number of C;

[0171] n: in the daughter ion 13 The number of C;

[0172] I: Peak area corresponding to the parent ion P from 0→p and the daughter ion D from 0→d;

[0173] k = 0.11 (in nature) 13 (Natural abundance of C);

[0174] mv≤pd.;

[0175] (8) Deconvolution of citric acid

[0176] 13 C glucose will produce 13 C-pyruvate enters the TCA cycle via the pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PC) pathways. Citrate (Cit) is a symmetrical molecule, but it contains a prochiral center and can be distinguished by the stereochemical characteristics of tricarboxylic acid cycle enzymes (e.g., Figure 2(As shown); Acetyl-CoA (AcCOA) provides C4 and C5 of citrate, and oxaloacetate (OAA) provides C1, 2, 3, and 6. If all the C4 and C5 are present at positions 4 and 5, then... 13 C-labeled Cit family (Cit) a,d,h,f,i,j ,like Figure 3 As shown in the diagram, carbon atoms generated by the PDH pathway can be resolved. Similarly, carbon atoms generated by the PC pathway can also be resolved by... 13 C-labeled Cit family (Cit) c,h )express;

[0177] In the fragmentation mode, removing C1, C6 or C5, C6 of Cit yields daughter ions, because these two carbon atoms contain information from the PC and PDH pathways. Deconvolving all daughter ions that have lost two carbon atoms yields metabolic flux information for the Cit family.

[0178] Without 13 The C-marked Cit is M, with 1 to 6 _ 13 The C-labeled Cit values ​​are represented as [M+2], [M+3], [M+4], [M+5], and [M+6], respectively. The parent / daughter combination assignments for individual [M+2] to [M+6] isotope groups are as follows:

[0179] There are two ways to generate Cit[M+2] with two tags: one is from the PDH pathway. a Secondly, the Cit generated from the PDH pathway in the second TCA cycle. a Clan and Cit b Cit a Fragments of the group can produce equal amounts of 193 / 68 and 193 / 69 daughter ions, Cit b Fragments of the group can produce 3 / 4 of the 193 / 68 daughter ions and 1 / 4 of the 193 / 69 daughter ions, from which we can derive the equation describing the isotopic composition of the parent / daughter combination:

[0180]

[0181]

[0182] Equations 1 and 2 can be solved to find:

[0183] Cit a = 3 * 193 / 69 - 193 / 68 (Equation 3)

[0184] Cit b = 2*(193 / 68-193 / 69) (Equation 4)

[0185] Similarly, we can conclude that:

[0186] Cit c = 3*194 / 69 - 5*194 / 68 - 5*194 / 70 (Equation 5)

[0187] Cit d = 4*194 / 68 - 4*194 / 69 + 12*194 / 70 (Equation 6)

[0188] Cit e = 2*194 / 68 - 2*194 / 69 - 6*194 / 70 (Equation 7)

[0189] Cit f = 2*195 / 70 + 2*195 / 69 (Equation 8)

[0190] Cit g = 9 * 195 / 69 - 195 / 70 (Equation 9)

[0191]

[0192]

[0193] Cit j =197 / 71 (Equation 12);

[0194] (9) Correction of isocitrate dehydrogenase

[0195] The deconvolution process assumes a direct flow of carbon from Cit to α-ketoglutarate (αKG). However, due to the presence of isocitrate dehydrogenase (ICDH), this reaction is reversible, and αKG can also be converted back to Cit. This reverse flux affects the isotopic labeling pattern of Cit. This ICDH reverse flux only affects C6-containing... 13 If the C-labeled Cit is unaffected, while the C4 and C5 labels are unaffected, then the correlation of this flux is with all [4,5- 13 C]Cit family (∑Cit) a Cit f Cit i Cit h Cit d Cit j ) and [1,2- 13 The sum of C2]AcCOA is directly proportional, defined as Φ AcCit (Isotope steady-state relationship):

[0196]

[0197] The following equations use ΦAcCit to correct the Q1 / Q3 segments of each Cit to eliminate the reverse ICDH flux and remove the effects of the reverse flux:

[0198]

[0199]

[0200]

[0201] 195 / 70 c =195 / 70*196 / 70(1-Φ AcCit )+195 / 69(1-Φ AcCit (Equation 17)

[0202]

[0203] 194 / 70 c =194 / 70-195 / 69(1-Φ) AcCit (Equation 19)

[0204] 194 / 69 c =194 / 69-195 / 69(1-Φ) AcCit )+194 / 68(1-Φ AcCit (Equation 20)

[0205]

[0206] 193 / 69 c =193 / 69-194 / 68(1-Φ) AcCit (Equation 22)

[0207] 193 / 68 c =193 / 68-194 / 68(1-Φ) AcCit )+(193 / 68-193 / 69)(1-Φ AcCit (Equation 23)

[0208] (10) Mass Isotope Analysis Distribution - Calculation of Isotopic Enrichment of Acetyl-CoA and Oxaloacetate

[0209] If pyruvate and AcCOA enrichment is known, the relative contributions of glucose oxidation and β-oxidation to AcCOA used by citrate synthase (CS) can be determined. In formal experiments, it may be difficult for researchers to directly measure the fractional enrichment of AcCOA and OAA in the mitochondrial matrix pool. This invention can solve this problem through mass isotope distribution analysis (MIDA).

[0210] For the reaction A + B → AB, if both substrates are partially enriched (FE) A* and FE B* ), then MIDA will determine the enrichment of the two precursors, even in the presence of dilution from externally unlabeled products, the partial enrichment (FE) of the matrix is ​​defined as:

[0211]

[0212]

[0213] (*) indicates the presence of a measurable label.

[0214] (A+A * ) and (B+B * The reaction will produce AB + AB. * +A * B+A * B * =1.

[0215] If unlabeled contaminant A'B' exists, then AB + AB * +A * B+A * B * +A'B' = 1.

[0216] Generate dual-labeled products (D A*B* ) and single-labeled substrate (S A*B and S AB* The probability of forming a product is determined by the fractional enrichment of the product:

[0217]

[0218]

[0219]

[0220] Equations 29 and 30 below describe the ratio of single-labeled products to dual-labeled products:

[0221]

[0222]

[0223] Solving the above equation yields the equation for FE:

[0224]

[0225]

[0226] Cit is formed in the mitochondrial matrix by condensing AcCOA and OAA using CS. Mass spectrometry can evaluate individual molecules, so D can be determined from the deconvolution of isotope families. A*B* S A*B and S AB* (See deconvolution of citrate isotopes), the inventors discovered several possible methods to calculate labeled AcCOA (FE) in the mitochondrial matrix. A* ) and marked OAA (FE) B* The partial enrichment of OAA and AcCOA. In summary, the steady-state enrichment of OAA and AcCOA can be calculated by analyzing the enrichment of several citrate isotope isomers, since any given citrate isotope isomer is a result of the enrichment of OAA and AcCOA, for example, [U- 13 C6]Cit is [U- 13 C4]OAA and [1,2- 13 The product of C2]AcCOA;

[0227] [1,2- 13 The calculation method for C2]AcCOA is as follows:

[0228]

[0229] 13 The equation for calculating OAA with C-label is as follows:

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] (11) Calculation of metabolic flux ratio based on isotopic steady state

[0236] Steady-state isotope analysis can determine whether there is a significant net inflow or exchange of unlabeled metabolites between successive or tandem metabolic reactions. One or more metabolic pathways may involve enzymatic reactions that produce products; for example, the formation of AcCOA primarily results from PDH or the β-oxidation of fatty acids or certain amino acids. If one pathway can be selectively labeled (e.g., pyruvate dehydrogenation or pyruvate carboxylation), the relative source of carbon inflow can be determined. Differential equations can be used to describe the rate of change in substrate-labeled metabolite enrichment due to metabolic inflow (inflow rate minus efflow rate). For generalized reactions:

[0237]

[0238] Where A is the initial substrate converted to product B by enzyme E1, and then B is converted to C by enzyme E2, the general equation is:

[0239]

[0240] Under metabolic and isotopic stability conditions 13 The change in C concentration over time is defined as zero. Therefore, the pathway from E1 to E2 with respect to (Φ 1→2 The solution can be obtained in such a way that the relative contribution of the input to the output is equal to the enrichment of the product on its precursor.

[0241]

[0242] If V E1 If V is the only path that helps generate B, then V E1 / V E2 The value will approach 1. However, this steady-state isotope analysis can only determine whether there is a significant net entry or exchange of unlabeled metabolites between sequential or tandem metabolic reactions (entry and exit are in equilibrium), and a value less than 1 indicates unlabeled input from another source. In simpler terms, Φ AB This refers to the relative contribution of substrate A to the pathway leading to product B, i.e., the metabolic rate ratio, while 1-Φ AB This indicates an unlabeled input to that pathway. It's important to note that this analysis cannot distinguish between reaction-replacement and exchange reactions, nor can it identify carbon losses from reaction-replacement.

[0243] Example 2

[0244] The aforementioned method for precise metabolic flux analysis of the TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry was used to explore the ratio of rat muscle metabolic substrate preference to metabolic rate under insulin stimulation. PDH / V CS The correlation between them is determined by the following steps:

[0245] (1) Rat feeding

[0246] Male Sprague-Dawley rats (250g each, 3 rats per cage) were housed in two groups (control group and insulin-resistant group) and fed a normal diet or a high-fat diet for 3 consecutive weeks. Then, all rats underwent surgery under general isoflurane anesthesia to place polyethylene catheters in the common carotid artery (PE50 catheter) and jugular vein (PE90 catheter), and were housed individually. For the following week, the rats were fed their respective diets before the studies were conducted. All in vivo studies were performed after a 16-hour overnight fast. At the end of each study, the rats were euthanized by intravenous administration of pentobarbital.

[0247] (2) [U- 13 C6] Glucose tracer in vivo enrichment experiment

[0248] In all studies, isotope tracers ([U- 13 [C6] Glucose was infused via a catheter placed in the carotid artery one week prior, with blood drawn from a catheter in the jugular vein. All studies began one hour after catheter connection to minimize any impact of stress response on the assessed physiology in the rats.

[0249] To measure V under basal fasting conditions PDH / V CS Flux, infusion of [U-] into rats 13 [C6] glucose (initially 3 mg / [Kg-min], continued for 5 minutes, followed by a continuous infusion rate of 1 mg / [Kg-min]), was administered for a total of 120 minutes. The rats were then sacrificed, and the skeletal muscle was rapidly frozen in situ using metal clamps pre-cooled in liquid nitrogen.

[0250] To measure V under insulin stimulation PDH / V CS The rats were administered a standard insulin bolus of 40 mU / kg, followed by an insulin infusion at a rate of 4.0 mU / (kg-min), simultaneously via a variable infusion [U- 13 C6] Glucose was used to maintain normal blood glucose levels. Rats were sacrificed 120 minutes after a total infusion, and skeletal muscle was flash-frozen in situ using metal clamps pre-cooled in liquid nitrogen.

[0251] (3) Preparation of liquid chromatography-mass spectrometry samples

[0252] Weigh 100 mg of rat skeletal muscle sample and add 0.1 mmol of taurine (internal standard). Homogenize the sample in 500 μL of ice-cold methanol using TissueLyser and filter through a Nanosep filter.

[0253] (4) Liquid chromatography-mass spectrometry analysis

[0254] A single standard was dissolved in 50% 1M HCl and 50% methanol to prepare a 5mM stock solution. On the day of the experiment, a mixed standard was prepared by serially diluting the mixed standard with ultrapure water containing 0.1 mmol taurine (internal standard) to 200 / 100 / 50 / 25 / 12.5 / 6.75 / 3.125 / 1.5625 / 0.78125 μM to plot a standard curve. After the analysis of 10 samples, the mixed sample wells were analyzed once. Mass spectrometry in multiple reaction monitoring mode (MRM mode) was used to monitor the stable isotopes. 13 Mass spectrometry analysis was performed on the cell sample extract cultured in C-labeled medium. Liquid chromatography-mass spectrometry analysis of the target metabolites was performed in a Waters Acquity UPLC system under the following chromatographic conditions:

[0255] Chromatographic column: ACQUITY UPLC@HSS T3 column, 2.1mm×100mm, 1.8μm;

[0256] Mobile phases: Phase A: 0.1% formic acid aqueous solution, and Phase B: 0.1% formic acid acetonitrile solution;

[0257] The liquid phase gradient program was: 0.0–0.5 min, 99% A; 0.5–4.0 min, 99%–95% A; 4.01–6.0 min, 99% A.

[0258] Column temperature: 35℃;

[0259] Sample cell temperature: 4℃;

[0260] Mobile phase flow rate: 0.4 mL / min;

[0261] Injection volume: 1 μL;

[0262] Mass spectrometry detection was performed using a Xevo TQ-S mass spectrometer in electrospray ionization (ESI) negative ion detection mode. The source parameters for the mass spectrometry analysis method are as follows:

[0263] Capillary voltage in negative ion mode: 2.50kV;

[0264] The cone voltage and collision voltage settings depend on the specific MRM channel for each metabolite (as shown in Table 1 below);

[0265] Desolventizing gas flow rate: 1000 L / h;

[0266] Temperature: 550℃;

[0267] Conical orifice gas flow rate: 150 L / h;

[0268] Atomizer setting: 7.0 Bar;

[0269] In MRM mode, parent / daughter ions are screened simultaneously to collect data. All of the above operations are controlled by MassLynx software.

[0270] Table 1

[0271]

[0272]

[0273]

[0274] (5) Background correction of liquid chromatography-mass data

[0275] Export peak area data from MassLynx software, using each 13 The peak area of ​​the C-labeled sample minus the ordinary 12 The peak area of ​​sample C is the peak area of ​​each sample after background correction.

[0276] (6) Natural abundance correction

[0277] In order to remove from nature 13 The influence of C requires the measured values ​​to be... 13 C was used to correct for the natural abundance to obtain the experimentally added... 13 C abundance, natural abundance adjusted by 1.1%, corrected isotope matrix I' (Pm，Dn) This is to explain the presence of naturally abundant carbon in each possible combination of mother / daughter ions in the matrix. (Pm，Dn) :

[0278] I′(P m D n )=I(P m D n )*(1+K(pm))-I(P m-1 D n )*k((pd)-(mn-1))-I(P m-1 D n-1 )*k(d-(n-1))

[0279] p: Total number of carbons in the parent ion;

[0280] d: The total number of carbons in the daughter ions;

[0281] m: in the parent ion 13 The number of C;

[0282] n: in the daughter ion 13 The number of C;

[0283] I: Peak area corresponding to the parent ion P from 0→p and the daughter ion D from 0→d;

[0284] k = 0.11 (in nature) 13 (Natural abundance of C);

[0285] mv≤pd.;

[0286] (7) Metabolic flux ratio V PDH / V CS calculate

[0287] PDH will [U- 13 C3]pyruvate is converted to [1,2- 13 C2]AcCOA, the latter can be further oxidized by citrate synthase (CS) (e.g. Figure 4 ). [1,2- 13 The variation of C2]AcCOA over time and its mass balance relationship are described by the following equations 41 and 42. Since [U- 13 C3]PEP better represents the true enrichment of glycolytic precursors, and we use [U-] in all the following equations. 13 C3]PEP enrichment replaced [U- 13 C3]pyruvate.

[0288]

[0289] V PDH +V βOX =V CS (Equation 42)

[0290] Under steady-state conditions, Equation 1 can be further simplified and V can be solved. PDH / V CS Obtain the relative flux (Φ) from PDH to CS PAc (Equation 3). [1,2- 13 The enrichment in C2]AcCoA can be calculated as described in "Calculation of AcCoA Enrichment", while [U- 13 C3]PEP can be measured directly.

[0291]

[0292] Although the real V PDH / V CS The calculation requires comparing intracellular components composed of [U- 13 C6] Glucose tracer enriched [U- 13 C3]PEP and [1,2- 13 The ratio of [C2]AcCOA is difficult to measure because these metabolites degrade rapidly in vitro. Under steady-state conditions, it is assumed that [U-] 13 C3]Pyruvic acid and [U-13 [C3] alanine balance, and it is assumed that [1,2- 13 C2]AcCOA and [4,5- 13 [C2] Glutamate balance. Alanine and glutamate are more stable in vitro than their respective counterparts and are better suited for reliable and consistent measurements of their intracellular enrichment. Therefore, V PDH / V CS Flux was measured as the concentration of [4,5-] in skeletal muscle 2 hours after infusion of [13C6] glucose. 13 C2] glutamic acid and [U- 13 The ratio of C3] alanine.

[0293]

[0294] (8) Regarding the speed ratio V PDH / V CS Results Analysis

[0295] In metabolic pathways, the V1 / V2 flux ratio, which is related to changes in the metabolic pathway, is calculated. This ratio serves as an important indicator for assessing the mechanism and impact of the analyte on the metabolic pathway. By changing the control conditions and observing the changes in the V1 / V2 ratio, we can determine whether the analyte affects the pathway, how it affects the pathway, and the specific changes it causes. Metabolic rate ratio V PDH / V CS This is a typical example, reflecting the proportion of glucose oxidation in mitochondria relative to the total oxidation of mitochondrial matter.

[0296] like Figure 5 As shown, to verify whether mitochondrial substrate preference changes in high-fat-induced insulin resistance rats, the inventors injected rats with [U- 13 After C6] glucose, [4,5- 13 C2] glutamic acid and [U- 13 The amount of C3] alanine produced is used to characterize the metabolic flux (V) of the PDH pathway. PDH ) and the metabolic flux that produces citric acid (V CS ), and V PDH / V CS The ratio reflects the proportion of glucose oxidation to total mitochondrial oxidation. In both normal and insulin-resistant rat skeletal muscle, under insulin stimulation, V... PDH / V CS The ratios were all significantly elevated, although the proportion of elevation was reduced in insulin-resistant rats. This indicates that even in insulin-resistant rats, the metabolic pathways of their muscle tissue can still flexibly utilize glucose.

[0297] Example 3

[0298] The proposed method for precise metabolic flux analysis of the TCA cycle, based on isotope tracing technology combined with high-resolution mass spectrometry, is used to explore changes in the target metabolic pathway and the metabolic rate ratio V. PC / V CS The correlation between them is determined by the following steps:

[0299] (1) Preparation of culture medium:

[0300] The method for preparing sugar-free culture medium is as follows: add 0.2% BSA, 24mM sodium bicarbonate, and 10mM HEPES to DMEM medium (sigma-D5030), mix well with a magnetic stir bar, and adjust the pH to 7.4.

[0301] The preparation method of culture media containing different concentrations of glucose (2.5, 5, 7 or 9 mM) is as follows: 0.2% BSA, 24 mM sodium bicarbonate, 10 mM HEPES, 4 mM glutamine, 0.05 mM pyruvate and 0.45 mM lactate are added to DMEM medium (sigma-D5030), and finally 2.5, 5, 7 or 9 mM glucose are added respectively. The mixture is stirred with a magnetic stir bar and the pH is adjusted to 7.4.

[0302] The prepared culture medium was filtered through a 0.22 μM filter membrane, and 0.1% penicillin-streptomycin mixture was added. It was then stored at 4°C.

[0303] Contains different concentrations 13 The preparation method for C-labeled glucose (2.5, 5, 7, or 9 mM) culture medium is as follows: Add 0.2% BSA, 24 mM sodium bicarbonate, 10 mM HEPES, 4 mM glutamine, 0.05 mM pyruvate, and 0.45 mM lactate to DMEM medium (sigma-D5030). Finally, add 2.5, 5, 7, or 9 mM of [U-] glucose. 13 C6] glucose, mix well with a magnetic stir bar, and adjust the pH to 7.4;

[0304] The prepared culture medium was filtered through a 0.22 μM filter membrane and 0.1% penicillin-streptomycin mixture was added. It was then stored at 4°C.

[0305] (2) Culture of pancreatic islet cells (INS-1):

[0306] INS-1 cells were initially pre-incubated for 3 hours in DMEM medium (D5030, Sigma-Aldrich) supplemented with glucose (2.5, 5, 7, and 9 mM), glutamine (4 mM), pyruvate (0.05 mM), and lactate (0.45 mM) to reach metabolic stability before the addition of isotope tags. They were then washed with glucose-free DMEM medium, followed by [U-] 2.5, 5, 7, and 9 mM [U-] 13 C6] glucose was incubated for 3 hours to reach isotopic stability.

[0307] (3) Cell quenching:

[0308] Pre-chill 5mM HEPES, quenching buffer (20% methanol, 0.1% formic acid, 3mM sodium fluoride, 1mM phenylalanine, and 100μM ethylenediaminetetraacetic acid) and V-type 96-well plates on ice. After INS-1 cell culture, place the 96-well plates on ice, quickly aspirate the culture medium, add 2mL of pre-chilled (4℃) 5mM HEPES to each well to wash INS-1 cells, then aspirate them, add 150μL of ice-cold quenching buffer, scrape off the INS-1 cells from the culture medium with a spatula, and quickly transfer the INS-1 cells and quenching buffer mixture to the ice-cold 96-well plates. Seal the 96-well plates with aluminum foil, poke a hole in each well with a 20g needle, store them in a -80℃ freezer overnight, and then freeze-dry them.

[0309] (4) Preparation of liquid chromatography-mass spectrometry (LC-MS) samples:

[0310] Samples were prepared before mass spectrometry analysis. The centrifuge was pre-cooled to 4°C, and a V-type 96-well plate was placed on ice. The lyophilized INS-1 cell powder was resuspended in pre-cooled 50 μL of ultrapure water (containing 25 μM taurine), and the plate was centrifuged at 4000 rpm for 5 min. The supernatant was transferred to a new 96-well plate, and the above operation was repeated at least 3 times to ensure that there was no precipitate in the supernatant. The supernatant was then transferred to a sample vial. For quality control, a mixed sample well was created by drawing 2.50 μL from each well.

[0311] (5) Liquid chromatography-mass spectrometry analysis

[0312] A single standard was dissolved in 50% 1M HCl and 50% methanol to prepare a 5mM stock solution. On the day of the experiment, a mixed standard was prepared by serially diluting the mixed standard with ultrapure water containing 25μM taurine (internal standard) to 200 / 100 / 50 / 25 / 12.5 / 6.75 / 3.125 / 1.5625 / 0.78125μM to plot a standard curve. After the analysis of 10 samples, the mixed sample well was analyzed once. Mass spectrometry in multiple reaction monitoring mode (MRM mode) was used to monitor the stable isotopes. 13Mass spectrometry analysis was performed on the extracts of cells cultured in C-labeled medium. Liquid chromatography-mass spectrometry analysis of the target metabolites was performed in a Waters Acquity UPLC system under the following chromatographic conditions:

[0313] Chromatographic column: ACQUITY UPLC@HSS T3 column, 2.1mm×100mm, 1.8μm;

[0314] Mobile phases: Phase A: 0.1% formic acid aqueous solution, and Phase B: 0.1% formic acid acetonitrile solution;

[0315] The liquid phase gradient program was: 0.0–0.5 min, 99% A; 0.5–4.0 min, 99%–95% A; 4.01–6.0 min, 99% A.

[0316] Column temperature: 35℃;

[0317] Sample cell temperature: 4℃;

[0318] Mobile phase flow rate: 0.4 mL / min;

[0319] Injection volume: 1 μL;

[0320] Mass spectrometry detection was performed using a Xevo TQ-S mass spectrometer in electrospray ionization (ESI) negative ion detection mode. The source parameters for the mass spectrometry analysis method are as follows:

[0321] Capillary voltage in negative ion mode: 2.50kV;

[0322] The cone voltage and collision voltage settings depend on the specific MRM channel for each metabolite (as shown in Table 1 below);

[0323] Desolventizing gas flow rate: 1000 L / h;

[0324] Temperature: 550℃;

[0325] Conical orifice gas flow rate: 150 L / h;

[0326] Atomizer setting: 7.0 Bar;

[0327] In MRM mode, parent / daughter ions are screened simultaneously to collect data. All of the above operations are controlled by MassLynx software.

[0328] Table 1

[0329]

[0330]

[0331] (6) Background correction of liquid chromatography-mass data

[0332] Export peak area data from MassLynx software, using each 13 The peak area of ​​the C-labeled sample minus the ordinary 12 The peak area of ​​sample C is the peak area of ​​each sample after background correction.

[0333] (7) Natural abundance correction

[0334] In order to remove from nature 13 The influence of C requires the measured values ​​to be... 13 C was used to correct for the natural abundance to obtain the experimentally added... 13 C abundance, natural abundance adjusted by 1.1%, corrected isotope matrix I' (Pm，Dn) This is to explain the presence of naturally abundant carbon in each possible combination of mother / daughter ions in the matrix. (Pm，Dn) :

[0335] I′(P m D n )=I(P m D n )*(1+K(pm))-I(P m-1 D n )*k((pd)-(mn-1))-I(P m-1 D n-1 )*k(d-(n-1))

[0336] p: Total number of carbons in the parent ion;

[0337] d: The total number of carbons in the daughter ions;

[0338] m: in the parent ion 13 The number of C;

[0339] n: in the daughter ion 13 The number of C;

[0340] I: Peak area corresponding to the parent ion P from 0→p and the daughter ion D from 0→d;

[0341] k = 0.11 (in nature) 13 (Natural abundance of C);

[0342] mv≤pd.;

[0343] (8) Deconvolution of citric acid

[0344] 13 C glucose will produce 13C-pyruvate enters the TCA cycle via the pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PC) pathways. Citrate (Cit) is a symmetrical molecule, but it contains a prochiral center and can be distinguished by the stereochemical characteristics of tricarboxylic acid cycle enzymes (e.g., Figure 2 (As shown); Acetyl-CoA (AcCOA) provides C4 and C5 of citrate, and oxaloacetate (OAA) provides C1, 2, 3, and 6. If all the C4 and C5 are present at positions 4 and 5, then... 13 C-labeled Cit family (Cit) a,d,h,f,i,j ,like Figure 3 As shown in the diagram, carbon atoms generated by the PDH pathway can be resolved. Similarly, carbon atoms generated by the PC pathway can also be resolved by... 13 C-labeled Cit family (Cit) c,h )express;

[0345] In the fragmentation mode, removing C1, C6 or C5, C6 of Cit yields daughter ions, because these two carbon atoms contain information from the PC and PDH pathways. Deconvolving all daughter ions that have lost two carbon atoms yields metabolic flux information for the Cit family.

[0346] Without 13 The C-marked Cit is M, with 1 to 6 _ 13 The C-labeled Cit values ​​are represented as [M+2], [M+3], [M+4], [M+5], and [M+6], respectively. The parent / daughter combination assignments for individual [M+2] to [M+6] isotope groups are as follows:

[0347] There are two ways to generate Cit[M+2] with two tags: one is from the PDH pathway. a Secondly, the Cit generated from the PDH pathway in the second TCA cycle. a Clan and Cit b Clan, Cit a Fragments of the group can produce equal amounts of 193 / 68 and 193 / 69 daughter ions, Cit b Fragments of the group can produce 3 / 4 of the 193 / 68 daughter ions and 1 / 4 of the 193 / 69 daughter ions, from which we can derive the equation describing the isotopic composition of the parent / daughter combination:

[0348]

[0349]

[0350] Equations 1 and 2 can be solved to find:

[0351] Cit a = 3 * 193 / 69 - 193 / 68 (Equation 3)

[0352] Cit b = 2*(193 / 68-193 / 69) (Equation 4)

[0353] Similarly, we can conclude that:

[0354] Cit c = 3*194 / 69 - 5*194 / 68 - 5*194 / 70 (Equation 5)

[0355] Cit d = 4*194 / 68 - 4*194 / 69 + 12*194 / 70 (Equation 6)

[0356] Cit e = 2*194 / 68 - 2*194 / 69 - 6*194 / 70 (Equation 7)

[0357] Cit f = 2*195 / 70 + 2*195 / 69 (Equation 8)

[0358] Cit g = 9 * 195 / 69 - 195 / 70 (Equation 9)

[0359]

[0360]

[0361] Cit j =197 / 71 (Equation 12);

[0362] (9) Correction of isocitrate dehydrogenase

[0363] The deconvolution process assumes a direct flow of carbon from Cit to α-ketoglutarate (αKG). However, due to the presence of isocitrate dehydrogenase (ICDH), this reaction is reversible, and αKG can also be converted back to Cit. This reverse flux affects the isotopic labeling pattern of Cit. This ICDH reverse flux only affects C6-containing... 13 If the C-labeled Cit is unaffected, while the C4 and C5 labels are unaffected, then the correlation of this flux is with all [4,5- 13 C]Cit family (∑Cit) a Cit f Cit i Cit h Cit d Cit j ) and [1,2- 13 The sum of C2]AcCOA is directly proportional, defined as Φ AcCit(Isotope steady-state relationship):

[0364]

[0365] The following equations use ΦAcCit to correct the Q1 / Q3 segments of each Cit to eliminate the reverse ICDH flux and remove the effects of the reverse flux:

[0366]

[0367]

[0368]

[0369] 195 / 70 c =195 / 70*196 / 70(1-Φ AcCit )+195 / 69(1-Φ AcCit (Equation 17)

[0370]

[0371] 194 / 70 c =194 / 70-195 / 69(1-Φ) AcCit (Equation 19)

[0372] 194 / 69 c =194 / 69-195 / 69(1-Φ) AcCit )+194 / 68(1-Φ AcCit (Equation 20)

[0373]

[0374] 193 / 69 c =193 / 69-194 / 68(1-Φ) AcCit (Equation 22)

[0375] 193 / 68 c =193 / 68-194 / 68(1-Φ) AcCit )+(193 / 68-193 / 69)(1-Φ AcCit (Equation 23)

[0376] (10) Mass Isotope Analysis Distribution - Calculation of Isotopic Enrichment of Acetyl-CoA and Oxaloacetate

[0377] If pyruvate and AcCOA enrichment is known, the relative contributions of glucose oxidation and β-oxidation to AcCOA used by citrate synthase (CS) can be determined. In formal experiments, it may be difficult for researchers to directly measure the fractional enrichment of AcCOA and OAA in the mitochondrial matrix pool. This invention can solve this problem through mass isotope distribution analysis (MIDA).

[0378] For the reaction A + B → AB, if both substrates are partially enriched (FE) A* and FE B* ), then MIDA will determine the enrichment of the two precursors, even in the presence of dilution from externally unlabeled products, the partial enrichment (FE) of the matrix is ​​defined as:

[0379]

[0380]

[0381] (*) indicates the presence of a measurable label.

[0382] (A+A * ) and (B+B * The reaction will produce AB + AB. * +A * B+A * B * =1.

[0383] If unlabeled contaminant A'B' exists, then AB + AB * +A * B+A * B * +A'B' = 1.

[0384] Generate dual-labeled products (D A*B* ) and single-labeled substrate (S A*B and S AB* The probability of forming a product is determined by the fractional enrichment of the product:

[0385]

[0386]

[0387]

[0388] Equations 29 and 30 below describe the ratio of single-labeled products to dual-labeled products:

[0389]

[0390]

[0391] Solving the above equation yields the equation for FE:

[0392]

[0393]

[0394] Cit is formed in the mitochondrial matrix by condensing AcCOA and OAA using CS. Mass spectrometry can evaluate individual molecules, so D can be determined from the deconvolution of isotope families. A*B* S A*B and S AB* (See deconvolution of citrate isotopes), the inventors discovered several possible methods to calculate labeled AcCOA (FE) in the mitochondrial matrix. A* ) and marked OAA (FE) B* The partial enrichment of OAA and AcCOA. In summary, the steady-state enrichment of OAA and AcCOA can be calculated by analyzing the enrichment of several citrate isotope isomers, since any given citrate isotope isomer is a result of the enrichment of OAA and AcCOA, for example, [U- 13 C6]Cit is [U- 13 C4]OAA and [1,2- 13 The product of C2]AcCOA;

[0395] [1,2- 13 The calculation method for C2]AcCOA is as follows:

[0396]

[0397] 13 The equation for calculating OAA with C-label is as follows:

[0398]

[0399]

[0400]

[0401]

[0402]

[0403] (11) Calculation of metabolic flux ratio based on isotopic steady state

[0404] Steady-state isotope analysis can determine whether there is a significant net inflow or exchange of unlabeled metabolites between successive or tandem metabolic reactions. One or more metabolic pathways may involve enzymatic reactions that produce products; for example, AcCOA formation primarily results from PDH or the β-oxidation of fatty acids or certain amino acids. If one pathway can be selectively labeled (e.g., pyruvate dehydrogenation or pyruvate carboxylation), the relative source of carbon inflow can be determined. Differential equations can be used to describe the rate of change in substrate-labeled metabolite enrichment due to metabolic inflow (inflow rate minus efflow rate). For generalized reactions:

[0405]

[0406] Where A is the initial substrate converted to product B by enzyme E1, and then B is converted to C by enzyme E2, the general equation is:

[0407]

[0408] Under metabolic and isotopic stability conditions 13 The change in C concentration over time is defined as zero. Therefore, the pathway from E1 to E2 with respect to (Φ 1→2 The solution can be obtained in such a way that the relative contribution of the input to the output is equal to the enrichment of the product on its precursor.

[0409]

[0410] If V E1 If V is the only path that helps generate B, then V E1 / V E2 The value will approach 1. However, this steady-state isotope analysis can only determine whether there is a significant net entry or exchange of unlabeled metabolites between sequential or tandem metabolic reactions (entry and exit are in equilibrium), and a value less than 1 indicates unlabeled input from another source. In simpler terms, Φ AB This refers to the relative contribution of substrate A to the pathway leading to product B, i.e., the metabolic rate ratio, while 1-Φ AB This indicates an unlabeled input to that pathway. It's important to note that this analysis cannot distinguish between reaction-replacement and exchange reactions, nor can it identify carbon losses from reaction-replacement.

[0411] (12) Metabolic flux ratio V PC / V CS calculate

[0412] Pyruvate decarboxylase (PC) is a mitochondrial enzyme that converts pyruvate to oxaloacetate (OAA) (e.g. Figure 6 From [U- 13 C3]Pyruvate synthesis [(1,2,3)(2,3,4)- 13The relative rate of C3]OAA can be determined similarly to that of PDH. Under metabolic homeostasis, OAA and malate can be considered as a measure of exchange due to the rapid exchange relative to the TCA cycle flux. Therefore, both phosphoenolpyruvate carboxylkinase (PEPCK) and malate enzyme (ME) must be considered as consuming [(1,2,3)(2,3,4)- 13 The reaction of C3]OAA. [(1,2,3)(2,3,4)- 13 The change of C3]OAA over time and the mass balance relationship are described by equations 4 and 5, respectively.

[0413] V PC +V CS =V CS +V PEPCK +V ME →V PC =V PEPCK +V ME (Equation 44)

[0414]

[0415] Under steady-state conditions, equations 4 and 5 can be further simplified and V can be solved. PC / V CS Obtain the relative flux (Φ) from PC to CS PO (Equations 46 and 47).

[0416]

[0417]

[0418] (13) Assay of insulin secretion in INS-1 cells: Insulin concentrations in culture medium extracts were measured using the Rat High Range Insulin ELISA Kit (ALPCO) according to the manufacturer’s instructions. All culture medium extracts were normalized to cellular protein concentrations using the Solarbio-BCA Protein Assay Kit.

[0419] (14) Speed ​​ratio V PC / V CS Link with insulin secretion in INS-1 cells

[0420] Through experimental verification, the inventors discovered that the metabolic flux rate is higher than V. PC / V CS It is closely related to glucose concentration and insulin secretion, and can be used to characterize cellular glucose metabolism and insulin secretion.

[0421] As a model system, pancreatic β-cells link glucose-dependent metabolic changes with the functional output of insulin secretion. The clonal insulinoma cell line INS-1 has become a model cell in many published experiments, and insulin release shows a linear correlation within the physiological glucose concentration range (2.5–9 mM) (e.g., ...). Figure 7 A). The inventors attempted to link changes in metabolic flux ratios in metabolomics analysis with insulin secretion. Specifically, in mitochondrial metabolic homeostasis, only changes in glucose concentration were correlated with the relative flux from PC to CS (Φ). PO Proportional changes (e.g.) Figure 7 B). This indicates that the metabolic flux rate is higher than V. PC / V CS It can characterize the relationship between insulin secretion and glucose concentration, and has potential applications in characterizing cellular glucose metabolism and insulin secretion.

Claims

1. A method for precise metabolic flux analysis of the TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry, characterized in that, Includes the following steps: S1: Cell Culture: After starving the cells for 1–4 hours, culture them in the first culture medium for 1–6 hours to reach cellular metabolic homeostasis, and then use a medium containing isotopes. 13 The second culture medium in C marks cells to reach isotopic homeostasis in 1–6 h; if the cells are hepatocytes, the glycogen substrates are 1–20 mM lactate and 1–20 mM pyruvate; if the cells are pancreatic islet cells, the glycogen substrates are 1–20 mM glucose and 1–20 mM glutamine, and the pH is adjusted to 6.8–7.

8. S2: Preparation of liquid mass samples: Collect cells with 50-200 μL of cell quenching agent and freeze-dry them, then rehydrate the frozen stem cells with 20-100 μL / well of rehydration solvent, and centrifuge to obtain the supernatant to prepare liquid mass samples. S3: Liquid chromatography-mass spectrometry (LC-MS) was used for liquid chromatography-mass spectrometry (LC-MS) analysis; S4: Liquid chromatography-mass analysis data processing; S41: Natural abundance correction; S42: Citric acid deconvolution; S43: Correction of isocitrate dehydrogenase; S44: The contents of acetyl-CoA and oxaloacetate were determined by mass isotope distribution analysis; S45: Calculation of metabolic flux ratio based on isotopic steady state; In the liquid chromatography-mass spectrometry (LC-MS) data processing described in S42, the fragment ion information is obtained by removing C1, C6, or C5, C6 from the parent ion of citrate (Cit). Since these two carbon atoms contain information from the pyruvate decarboxylase and pyruvate dehydrogenase pathways, deconvolution analysis is performed on all fragment ions to obtain the citrate family: Cit. a Cit b Cit c Cit d Cit e Cit f Cit g Cit h Cit i Cit j The metabolic flux information is calculated using the following formula: Cit a =3*193 / 69-193 / 68 Cit b =2*(193 / 68-193 / 69) Cit c =3*194 / 69-5*194 / 68-5*194 / 70 Cit d =4*194 / 68-4*194 / 69+12*194 / 70 Cit e =2*194 / 68-2*194 / 69-6*194 / 70 Cit f =2*195 / 70+2*195 / 69 Cit g =9*195 / 69-195 / 70 Cit j =197 / 71; The correction of isocitrate dehydrogenase described in S43, through [4,5- 13 C2]citric acid and [1,2- 13 The ratio of C2] acetyl-CoA is used to correct for the backflow of labeled carbon caused by the countercurrent of the isocitrate dehydrogenase reaction, making the citrate deconvolution result in S42 more accurate. The calculation formula is as follows: 195 / 70 c =195 / 70*196 / 70(1-Φ AcCit )+195 / 69(1-Φ AcCit ) 194 / 70 c =194 / 70-195 / 69(1-Φ AcCit ) 194 / 69 c =194 / 69-195 / 69(1-Φ AcCit )+194 / 68(1-Φ AcCit ) 193 / 69 c =193 / 69-194 / 68(1-Φ AcCit ) 193 / 68 c =193 / 68-194 / 68(1-Φ) AcCit )+(193 / 68-193 / 69)(1-Φ AcCit The contents of acetyl-CoA and oxaloacetate in S44 were determined by citric acid mass isotope distribution analysis, and the calculation formula is as follows: [1,2- 13 The calculation method for C2]AcCOA is as follows: 13 The equation for calculating OAA with C-label is as follows: The natural abundance correction in S41 is achieved by measuring the natural abundance. 13 C performs natural abundance correction to remove naturally occurring pollutants. 13 The effect of C, thus obtaining the results of the experiment. 13 C abundance is calculated using the following formula: I′(P m ,D n )=I(P m ,D n )*(1+K(p-m))-I(P m-1 ,D n )*k((p-d)-(m-n-1))-I(P m-1 ,D n-1 )*k(d-(n-1)) p: Total number of carbons in the parent ion; d: The total number of carbons in the daughter ions; m: in the parent ion 13 The number of C; n: in the daughter ion 13 The number of C; I: Peak area corresponding to the parent ion P from 0→p and the daughter ion D from 0→d; k = 0.11, in nature 13 Natural abundance of C; mv≤pd.

2. The method for precise metabolic flux analysis of the TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry as described in claim 1, characterized in that, The first culture medium in S1 is prepared by adding 0.1–2.0% BSA, 12–30 mM sodium bicarbonate, 5–15 mM HEPES, and 0–25 mM saccharogenic substrate to DMEM medium, mixing with a magnetic stir bar, adjusting the pH to 6.8–7.8, filtering through a 0.22 μM filter membrane, adding 0.1% penicillin-streptomycin mixture, and storing at 4°C. The second culture medium is prepared by adding 0.1–2% BSA, 12–30 mM sodium bicarbonate, 5–15 mM HEPES, and 0–25 mM saccharogenic substrate to DMEM medium. 13 The C-labeled saccharogenic substrate was mixed with a magnetic stir bar, the pH was adjusted to 6.8–7.8, filtered through a 0.22 μM filter membrane, and 0.1% penicillin-streptomycin mixture was added. The mixture was then stored at 4 °C.

3. The method for precise metabolic flux analysis of the TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry as described in claim 1, characterized in that, The cell quenching agent described in S2 is composed of 10–40% methanol, 0.1–1% formic acid, 0.5–5 mM sodium fluoride, 1–2 mM phenylalanine, and 50–150 μM ethylenediaminetetraacetic acid; the resolvent is a 2–100 μM taurine aqueous solution.

4. The method for precise metabolic flux analysis of the TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry as described in claim 1, characterized in that, The liquid chromatography-mass spectrometry (LC-MS) system described in S3 is the Waters Acquity UPLC system, and (1) the chromatographic conditions are: Chromatographic column: ACQUITY UPLC@HSS T3 column, 2.1mm×100mm, 1.8μm; Mobile phases: Phase A: 0.1% formic acid aqueous solution, and Phase B: 0.1% formic acid acetonitrile solution; The liquid phase gradient program was: 0.0–0.5 min, 99% A; 0.5–4.0 min, 99%–95% A; 4.01–6.0 min, 99% A. Column temperature: 20~55℃; Sample cell temperature: 4~10℃; Mobile phase flow rate: 0.2–0.5 mL / min; Injection volume: 1 μL; (2) Mass spectrometry detection was performed using a Xevo TQ-S mass spectrometer in negative ion detection mode with an electrospray ionization source. The source parameters for the mass spectrometry analysis method are as follows: Capillary voltage: 0.2~3.2kV; Desolventizing gas flow rate: 800~1200L / h; Temperature: 450~600℃; Conical orifice gas flow rate: 100~250L / h; Atomizer setting: 5-10 Bar.

5. The method for precise metabolic flux analysis of the TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry as described in claim 1, characterized in that, The metabolic flux ratio at steady state, as described in S45, is calculated by determining, through steady-state isotope analysis, whether there is a net inflow, outflow, or exchange of unlabeled metabolites between consecutive or tandem metabolic reactions, and by using differential equations to calculate the rate ratio between metabolic pathways: This describes the rate of change in the enrichment of substrate-labeled metabolites due to metabolic changes.

Images