CN-116183788-B - Method for analyzing TCA (ternary content addressable memory) cycle by combining isotope tracing technology with high-resolution mass spectrum

Abstract

The invention belongs to the field of biological analysis, and provides a method for analyzing TCA (ternary content addressable memory) circulation by combining an isotope tracing technology with high-resolution mass spectrometry. By optimizing mass spectrum parameters and a liquid chromatography method, one sample injection within 6min is realized, and 11 biomarkers related to TCA (ternary content addressable memory) circulation pathway metabolism are detected with high flux, high sensitivity and high separation degree. Meanwhile, the present invention innovatively uses LC-MS/MS to decipher 13 C's stepwise position-specific transfer from glucose to subsequent metabolites through glycolysis and TCA cycle, thereby calculating the integrated steady-state analysis of key metabolic rates (pyruvate dehydrogenase, β -oxidation, pyruvate carboxylase, isocitrate dehydrogenase and pyruvate cycle) for position-specific transfer of 13 C from sequential precursors to their products. This technique has broad applicability and can potentially characterize mitochondrial metabolism in any tissue or cell.

Inventors

• MA LINGJUN
• YU YIRAN
• LI WEI
• Mou yao
• JI JUNFU
• CHEN FANG
• HU XIAOSONG

Assignees

• 中国农业大学

Dates

Publication Date

20260508

Application Date

20230109

Claims (5)

1. 1. The method for analyzing the TCA cycle by combining the isotope tracking technology with the high-resolution mass spectrum is characterized by comprising the following steps of: S1, culturing cells, namely culturing the cells for 1-6 hours by using a first culture medium after starving the cells for 1-4 hours to reach a cell metabolic steady state, and marking the cells for 1-6 hours by using a second culture medium with an isotope 13 C to reach the cell isotopic steady state, wherein if the cells are liver cells, the substrates for glucose production are 1-20 mM lactic acid and 1-20 mM pyruvic acid, and if the cells are islet cells, the substrates for glucose production are 1-20 mM glucose and 1-20 mM glutamine, and the pH is adjusted to 6.8-7.8; S2, preparing a liquid sample, namely collecting cells by using 50-200 mu L of cell quenching agent, freeze-drying, re-dissolving the freeze-dried cells by using 20-100 mu L/hole of re-solvent, and centrifuging to obtain a supernatant to prepare the liquid sample; S3, adopting a liquid chromatography-mass spectrometer to perform liquid chromatography; s4, liquid quality analysis data processing; s41, natural abundance correction; s42, deconvolution of citric acid; S43, correcting isocitrate dehydrogenase; S44, measuring the contents of acetyl coenzyme A and oxaloacetic acid through mass isotope distribution analysis; s45, calculating a metabolic flow rate ratio based on isotope steady-state; The sub-ion fragment information in the liquid analysis data processing in S42 is obtained by cutting C1, C6 or C5, C6 of the citric acid Cit from the parent ion, and since the two carbon atoms contain the information from the pyruvate decarboxylase pathway and the pyruvate dehydrogenase pathway, the metabolic flux information of the citric acid group ：Cit a 、Cit b 、Cit c 、Cit d 、Cit e 、Cit f 、Cit g 、Cit h 、Cit i 、Cit j is obtained by deconvoluting all the sub-ion fragments, and the calculation formula is as follows: 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 in S43 corrects the marked carbon reflux phenomenon caused by the reaction countercurrent of isocitrate dehydrogenase by the ratio of [4,5- 13 C 2 ] citric acid to [1,2- 13 C 2 ] acetyl-CoA, so that the result of deconvolution of the citric acid in S42 is more accurate, and 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 ), and the content of acetyl coenzyme A and oxaloacetate is determined in S44 by citric acid mass isotope distribution analysis, wherein the calculation formula is as follows: The calculation method of [1,2- 13 C 2 ] AcCOA is as follows: 13 The equation for the C-labeled OAA is calculated as follows: The natural abundance correction in S41 corrects the measured 13 C natural abundance to remove the influence of 13 C in nature, so as to obtain the abundance of 13 C added in the experiment, and the calculation formula is as follows: 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 is the total number of carbons in the parent ion; d, total carbon in the sub-ions; m is the number of 13 C in the parent ion; n is the number of 13 C in the daughter ions; i, peak areas corresponding to the parent ion P from 0 to P and the child ion D from 0 to D; k=0.11, natural abundance of 13 C in nature; m-v≤p-d。
2. 2. The method for analyzing TCA cycle by combining isotope tracing technology with high-resolution mass spectrometry according to claim 1, wherein the preparation method of the first culture medium in S1 is characterized in that 0.1-2.0% BSA, 12-30 mM sodium bicarbonate and 5~15mM HEPES,0~25mM sugar substrate are added into a DMEM culture medium, uniformly mixed by a magnetic stirrer, pH is adjusted to 6.8-7.8, filtered by a filter membrane with 0.22 mu M, 0.1% of a mixture of green streptomycin is added, and the mixture is preserved at 4 ℃, the preparation method of the second culture medium is characterized in that 0.1-2% BSA, 12-30 mM sodium bicarbonate and 5~15mM HEPES,0~25mM 13 C labeled sugar substrate are added into the DMEM culture medium, uniformly mixed by a magnetic stirrer, pH is adjusted to 6.8-7.8, filtered by a filter membrane with 0.22 mu M, and preserved at 4 ℃.
3. 3. The method for analyzing TCA cycle by combining high-resolution mass spectrometry with accurate metabolic flow based on an isotope tracing technology according to claim 1, wherein the cell quencher in S2 is composed of 10-40% of methanol, 0.1-1% of formic acid, 0.5-5 mM of sodium fluoride, 1-2 mM of phenylalanine and 50-150 mu M of ethylenediamine tetraacetic acid, and the cosolvent is 2-100 mu M of taurine aqueous solution.
4. 4. The method for analyzing the TCA cycle by combining the isotope labeling technique with the accurate metabolic flux of the high-resolution mass spectrum according to claim 1, wherein the liquid chromatography-mass spectrometer in S3 is Waters Acquity UPLC systems, and the chromatographic conditions are: The chromatographic column is an ACQUITY UPLC@HSS T3 chromatographic column, 2.1mm multiplied by 100mm,1.8 μm; mobile phase A0.1% formic acid aqueous solution, and B0.1% formic acid acetonitrile solution; The liquid phase gradient procedure was: 0.0 to 0.5min,99% A, 0.5 to 4.0min,99% to 95% A, 4.01 to 6.0min,99% A; the column temperature is 20-55 ℃; the temperature of the sample cell is 4-10 ℃; the flow rate of the mobile phase is 0.2-0.5 mL/min; the sample injection amount is 1 mu L; (2) Mass spectrometry detection is carried out in an electrospray ionization source anion detection mode by using mass spectrometers Xevo TQ-S, and the source parameters of a mass spectrometry method are as follows: the capillary voltage is 0.2-3.2 kV; The flow rate of desolventizing gas is 800-1200L/h; the temperature is 450-600 ℃; the gas flow rate of the taper hole is 100-250L/h; The atomizer is arranged at 5-10 Bar.
5. 5. The method of claim 1, wherein the steady state metabolic flow rate ratio of the isotope in S45 is calculated by steady state isotope analysis to determine whether there is a net entry, anaplerosis, or exchange of unlabeled metabolites between consecutive or tandem metabolic reactions, and calculating the rate ratio between metabolic pathways using differential equations: to describe the rate of change of enrichment of the substrate-tagged metabolite due to metabolic changes.

Description

Method for analyzing TCA (ternary content addressable memory) cycle by combining isotope tracing technology with high-resolution mass spectrum Technical Field The invention belongs to the field of biological analysis, and relates to a method for analyzing TCA (ternary content addressable memory) circulation by combining an isotope tracing technology with high-resolution mass spectrometry. Background Since DNA was found, human cognition of genes and their protein products has been increasing and research in the biological field has been advancing. Over the last decade, with the continued prevalence of obesity and metabolic syndrome, there has been an increasing interest in metabonomics, which increasingly dominates over many areas of biology. Nowadays, more and more studies are being analyzed from a metabolic point of view. In general, with the continued advancement of metabolite measurement technology (especially mass spectrometry), metabonomics has profound research value. From bacteria to humans, the core of oxidative metabolism and anabolism surrounds the continuous enzymatic reaction of the citric acid cycle (TCA cycle), which is the cornerstone that constitutes the metabolism of the body. There are numerous alternate reaction nodes in mitochondria and the exchange, anaplerotic and decentralized reactions of metabolites at these nodes can complicate metabolic flux analysis. Metabolic activity can be quantified by using the mass flux per unit time, i.e., the metabolic flux, and measuring the metabolite concentration cannot be equivalent to the metabolite flux, and the current research method cannot accurately analyze the flux of the metabolite in the complex metabolic reaction. This may be because during metabolism, the accumulation of metabolites is not only due to an increase in yield but also due to a decrease in consumption. For example, when glucose is removed from yeast, although the glycolytic pathway inflow is reduced, the outflow is rapidly increased, the glycolytic flux is reduced, and at this time, the increase in the metabolite concentration in the outflow does not match the reduction in the glycolytic flux. Since metabolite levels and flux provide more comprehensive information, a comprehensive understanding of metabolism by researchers is best achieved by comprehensively analyzing both. The mass spectrometry method has the potential of simultaneously measuring labeled and unlabeled metabolites, improving sensitivity and analyzing single isotope isomers, and a comprehensive and direct metabolic flow platform is provided, so that the progressive position-specific transfer of mass isotope labels in continuous reactions of glycolysis and TCA (ternary content addressable memory) cycle is tracked and analyzed by an accurate metabolic flow method based on isotope tracing technology and high-resolution mass spectrometry. The technology mainly comprises the steps of directly deconvoluting citric acid mass spectrum information generated by 13 C marked glucose, lactic acid and other glycogenic substrates, so as to break the position-specific isotopic enrichment of acetyl coenzyme A, oxaloacetic acid, citrate and other TCA (ternary ammonium) circulatory metabolic intermediates in mitochondria. Meanwhile, it can calculate key metabolic rates (pyruvate dehydrogenase, beta-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and pyruvate circulation, etc.) of 13 C from the site-specific transfer of sequential precursors to their products to analyze the comprehensive steady state situation in cells. It is worth noting that the mass spectrum information deconvolution technology of TCA cycle intermediate metabolites such as citrate breaks through the important limitation that the prior isotope analysis technology can only measure the concentration of a target substance and cannot accurately analyze the specific flux. Meanwhile, the technology has wide applicability to any glucose oxidizing cell, and provides a new analysis method for analyzing the influence mechanism of the condition to be detected on the sugar metabolism and searching for an unknown metabolic pathway. Disclosure of Invention The quantitative calculation of metabolites in cellular mitochondria requires measurement of flux between metabolic reactions, and the exchange, anaplerotic and dispersive reactions of metabolites at these nodes can complicate isotope labeling due to the numerous staggered reaction nodes. However, current analytical methods do not accurately detect isotope flow in complex metabolic reactions. The inventors used LC-MS/MS to gradually analyze the position-specific transfer of isotopes (13 C) from glucose to subsequent metabolites in glycolysis and TCA cycle reactions, and by cleaving citrate isotope isomers to break up the cross metabolic flux, thereby comprehensively locating the precursor-to-product 13 C tag transfer changes in metabolic reactions, and accurately quantifying the exchange rate between oxidation, an