CN116183788A - A method for accurate metabolic flux analysis of TCA cycle based on isotope tracer technology combined with high-resolution mass spectrometry - Google Patents

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. At the same time, the invention innovatively uses LC-MS/MS to decipher ¹³ C from glucose to subsequent metabolites by glycolysis and stepwise site-specific transfer of the TCA cycleAnd calculate ¹³ Comprehensive steady state analysis of the key metabolic rates of site-specific transfer of sequential precursors to their products (pyruvate dehydrogenase, beta-oxidation, pyruvate carboxylase, isocitrate dehydrogenase and pyruvate circulation). This technique has broad applicability and can potentially characterize mitochondrial metabolism in any tissue or cell.

Description

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

Technical Field

The present invention belongs to the field of biological analysis and relates to a method for accurate metabolic flux analysis of TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry.

Background Art

Since the discovery of DNA, human knowledge of genes and their protein products has continued to increase, and research in the biological field has continued to advance. In the past decade, with the continued prevalence of obesity and metabolic syndrome, people's interest in metabolomics has increased, and metabolism has gradually become mainstream in many fields of biology. Nowadays, more and more studies are analyzed from the perspective of metabolism. In general, with the continuous advancement of metabolite measurement technology (especially mass spectrometry), metabolomics has far-reaching research value.

From bacteria to humans, the core of oxidative metabolism and anabolism revolves around the continuous enzymatic reactions of the citric acid cycle (TCA cycle), which is the cornerstone of the body's metabolism. There are many intertwined reaction nodes in mitochondria, and the exchange, replenishment and dispersion reactions of metabolites at these nodes complicate the analysis of metabolic flux. Metabolic activity can be quantified by the material flow per unit time, that is, metabolic flux. 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 the accumulation of metabolites in the metabolic process is not only due to increased production, but also due to reduced consumption. For example, when glucose is removed from yeast, although the influx of the glycolytic pathway decreases, the outflow will increase sharply and the glycolytic flux will decrease. At this time, the increase in metabolite concentration in the outflow does not match the decrease in glycolytic flux. Because metabolite levels and fluxes provide more comprehensive information, researchers' comprehensive understanding of metabolism is best achieved by comprehensively analyzing the two.

Mass spectrometry has the potential to measure both labeled and unlabeled metabolites, improve sensitivity, and analyze individual isotopomers. Here, we propose a comprehensive and direct metabolic flux platform to track and analyze the stepwise position-specific transfer of mass isotope labels in the sequential reactions of glycolysis and TCA cycle through a precise metabolic flux method based on isotope tracing technology combined with high-resolution mass spectrometry. Among them, this technology mainly includes direct deconvolution of the mass spectrum information of citrate produced by ¹³ C-labeled glucose, lactate and other glycogenic substrates, thereby deciphering the position-specific isotope enrichment of TCA cycle metabolic intermediates such as acetyl-CoA, oxaloacetate and citrate in mitochondria. At the same time, it can calculate the key metabolic rates of position-specific transfer of ¹³ C from sequential precursors to their products (pyruvate dehydrogenase, β-oxidation, pyruvate carboxylase, isocitrate dehydrogenase and pyruvate cycle, etc.) to analyze the comprehensive steady-state situation in cells. It is worth noting that the mass spectrometry information deconvolution technology of TCA cycle intermediate metabolites such as citrate has overcome the important limitation of previous isotope analysis technology that can only measure the concentration of the target but cannot accurately analyze its specific flux. At the same time, this technology has wide applicability to any glucose oxidizing cells, and it provides a new analytical method for analyzing the mechanism of the influence of the test conditions on sugar metabolism and exploring unknown metabolic pathways.

Summary of the invention

Technical problems to be solved: Quantitative calculation of metabolites in mitochondria requires the determination of fluxes between metabolic reactions. Due to the presence of numerous intersecting reaction nodes, the exchange, anaplerosis and dispersion reactions of metabolites at these nodes complicate isotope labeling. However, current analytical methods cannot accurately detect the isotope flow in complex metabolic reactions. The inventors used LC-MS/MS to gradually analyze the position-specific transfer of isotopes ( ^13C ) from glucose to subsequent metabolites in glycolysis and TCA cycle reactions, and deciphered the cross-linked metabolic fluxes by deciphering citrate isotopomers, thereby comprehensively locating the changes in the transfer of ^13C labels from precursors to products in metabolic reactions, and accurately quantifying the exchange rates between oxidation, anaplerosis, cycle reactions and metabolites in mitochondria.

Technical solution: The present invention provides a method for accurately analyzing TCA cycle metabolic flux based on isotope tracing technology combined with high-resolution mass spectrometry, comprising the following steps:

(1) Preparation of culture medium:

The method for preparing the sugar-free culture medium is as follows: 0.1-2.0% BSA, 12-30 mM sodium bicarbonate, and 5-15 mM HEPES are added to the DMEM culture medium (sigma-D5030), mixed with a magnetic stirrer, and the pH is adjusted to 6.8-7.8;

The preparation method of the high-glucose culture medium (first culture medium) is as follows: 0.1-2.0% BSA, 12-30 mM sodium bicarbonate, 5-15 mM HEPES, and 1-25 mM glycogen substrate (the glycogen substrate is glucose, lactic acid, pyruvic acid, or glutamine) are added to a DMEM culture medium (sigma-D5030), mixed with a magnetic stirrer, and the pH is adjusted to 6.8-7.8; wherein, if the cells are hepatocytes, the glycogen substrates are 1-20 mM lactic acid and 1-20 mM pyruvic acid, and if the cells are pancreatic islet cells, the glycogen substrates are 1-20 mM glucose and 1-20 mM glutamine;

The prepared culture medium was filtered with a 0.22 μM filter membrane, 0.1% penicillin-streptomycin mixture was added, and stored at 4°C;

The preparation method of ¹³ C-labeled high-glucose medium (second medium) is as follows: 0.1-2.0% BSA, 12-30 mM sodium bicarbonate, 5-15 mM HEPES, and 1-25 mM ¹³ C-labeled glycogenic substrate (the glycogenic substrate is [U- ¹³ C ₆ ]glucose, [4,5- ¹³ C ₂ ]glutamine or [U- ¹³ C ₃ ]lactic acid, [U- ¹³ C ₃ ]pyruvic acid) are added to DMEM medium (sigma-D5030), mixed with a magnetic stirrer, and the pH is adjusted to 6.8-7.8;

The prepared culture medium was filtered with a 0.22 μM filter membrane, and a 0.1% penicillin-streptomycin mixture was added and stored at 4°C;

(2) Cell culture:

When the cell density reached 80%, the cells were digested and seeded into a six-well plate with a complete medium (80% high-glucose medium + 10% FBS) at a density of 5×10 ⁵ cells/well. After the cells adhered to the wall and the density reached 80%, they were starved for 1 to 4 hours with a sugar-free medium. The cells were cultured with the first medium for 1 to 6 hours to achieve cell metabolic homeostasis, and then the cells were labeled with a second medium containing the isotope ¹³ C for 1 to 6 hours to achieve cell isotope homeostasis.

*The duration of the labeling incubation period varies from cell to cell, and preliminary quenching time course studies are performed to determine the point at which cells reach isotopic steady-state;

(3) Cell quenching:

Precool 5mM HEPES, quenching buffer (10-40% methanol, 0.1-1% formic acid, 0.5-5mM sodium fluoride, 1-2mM phenylalanine and 50-150μM ethylenediaminetetraacetic acid) and V-shaped 96-well plate on ice. After the cell culture is completed, put the 96-well plate on ice, quickly aspirate the culture medium, add 2mL of precooled (4°C) 5mM HEPES to each well to wash the cells, then aspirate it, add 150μL of ice-cold quenching buffer, scrape the cells on the culture medium with a scraper, and quickly transfer the cells and quenching buffer mixture to the 96-well plate on ice. Seal the 96-well plate with aluminum film, use a 20g needle to poke a hole in each well, store it in a -80°C freezer overnight, and then freeze-dry;

(4) Liquid sample preparation:

Prepare samples before mass spectrometry analysis. Precool the centrifuge to 4°C, place the V-shaped 96-well plate on ice, resuspend the freeze-dried cell powder in 50uL precooled ultrapure water (containing 2-100μM taurine), and centrifuge the 96-well plate at 4000r/min for 5min; transfer the supernatant to a new 96-well plate, repeat the above operation at least 3 times to ensure that there is no precipitate in the supernatant, and transfer it to a sample bottle. For quality control, create a mixed sample well by extracting 2-3μL from each well;

(5) Liquid chromatography-mass spectrometry analysis

A single standard was dissolved in 50% 1M HCl and 50% methanol to prepare a 5 mM stock solution. On the day of the experiment, ultrapure water containing 25 μM taurine (internal standard) was used to gradient dilute the mixed standard to 200/100/50/25/12.5/6.75/3.125/1.5625/0.78125 μM to draw a standard curve. After 10 samples were analyzed, the mixed sample well was analyzed once. The cell sample extract cultured in the stable isotope ¹³ C labeled culture medium was analyzed by mass spectrometry in the mass spectrometry multiple reaction monitoring mode (MRM mode). The liquid phase mass spectrometry analysis of the target metabolites was carried out on the Waters Acquity UPLC system. The chromatographic conditions were as follows:

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

Mobile phase: Phase A: 0.1% formic acid in water, and Phase B: 0.1% formic acid in acetonitrile;

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°C;

Mobile phase flow rate: 0.2～0.5mL/min;

Injection volume: 1 μL;

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

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

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

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

Temperature: 450～600℃;

Cone gas flow rate: 100～250L/h;

Atomizer setting: 5~10Bar;

Data were collected by screening parent/daughter ions simultaneously in MRM mode, all of which were controlled by MassLynx software;

Table 1

(6) Background correction of liquid quality data

The peak area data were exported in MassLynx software, and the peak area of each ¹³ C-labeled sample was subtracted from the peak area of the normal ¹² C sample to obtain the background-corrected peak area of each sample.

(7) Natural abundance correction

In order to remove the influence of ¹³ C in nature, the measured ¹³ C is corrected for natural abundance to obtain the ¹³ C abundance added in the experiment. The natural abundance is adjusted by 1.1%. The corrected isotope matrix I' _{(Pm, Dn)} is to explain the presence of natural abundance carbon for each possible parent/daughter ion combination in the matrix I _{(Pm, Dn)} :

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))

p: total number of carbons in the parent ion;

d: total number of carbons in the product ion;

m: the number of ¹³ C in the parent ion;

n: the number of ¹³ C in the product ion;

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

k = 0.11 (natural abundance of ¹³ C in nature);

m-v≤p-d.;

(8) Deconvolution of citric acid

^13C glucose will produce ^13C pyruvate, which enters the TCA cycle through the pyruvate dehydrogenase (PDH) pathway and the pyruvate decarboxylase (PC) pathway. Citric acid (Cit) is a symmetrical molecule, but it contains a prochiral center, which can be distinguished by the stereochemical characteristics of the tricarboxylic acid cycle enzymes (as shown in Figure 2); acetyl coenzyme A (AcCOA) provides C4 and C5 of citric acid, and oxaloacetate (OAA) provides C1, 2, 3 and 6. If all Cit groups with ^13C labels at positions 4 and 5 (Cit _{a, d, h, f, i, j} , as shown in Figure 3) can be measured, the carbon atoms produced by the PDH pathway can be analyzed. Similarly, the carbon atoms produced by PC are also represented by the ^13C -labeled Cit group (Cit _{c, h} );

In the fragmentation mode, the C1, C6 or C5, C6 of Cit are cut off to obtain the daughter ions. Since these two carbon atoms contain information from the PC pathway and the PDH pathway, the metabolic flow information of the Cit family can be obtained by deconvolving all the daughter ions that have lost the two carbon atoms.

Cit without ¹³ C label is M, Cit with 1 to 6 ¹³ C labels are represented as [M+2], [M+3], [M+4], [M+5], [M+6], and the mother/daughter combination assignments of the single [M+2] to [M+6] isotope groups are as follows:

There are two ways to produce Cit[M+2] with two labels: one is Cit _a produced from the PDH pathway, and the other is Cit _b produced from the PDH pathway in the second round of TCA cycle. The fragments of Cit _a family can produce equal amounts of 193/68 and 193/69 daughter ions, and the fragments of Cit _b family can produce 3/4 of 193/68 daughter ions and 1/4 of 193/69 daughter ions. From this, we can derive the equation describing the isotopic composition of the parent/daughter combination:

Equations 1 and 2 can be solved to obtain:

Ci _a =3*193/69-193/68 (Equation 3)

Cit _b = 2*(193/68-193/69) (Equation 4)

Similarly, we can conclude that:

Cit _c =3*194/69-5*194/68-5*194/70 (Equation 5)

Cit _d = 4*194/68-4*194/69+12*194/70 (Equation 6)

Cit _e =2*194/68-2*194/69-6*194/70 (Equation 7)

Cit _f = 2*195/70+2*195/69 (Equation 8)

Cit _g =9*195/69-195/70 (Equation 9)

Cit _j = 197/71 (Equation 12);

(9) Correction of isocitrate dehydrogenase

The deconvolution process assumes that carbon flows directly from Cit to α-ketoglutarate (αKG). However, due to the presence of isocitrate dehydrogenase (ICDH), the reaction is reversible and αKG is also converted back to Cit. This reverse flux will affect the isotope labeling pattern of Cit. This reverse flux of ICDH will only affect Cit containing ¹³ C labeling at C6, while the labels of C4 and C5 are not affected. The correlation of this flux is proportional to the sum of all [4,5- ¹³ C]Cit families (∑Cit _a , Cit _f , Cit _i , Cit _h , Cit _d , Cit _j ) and [1,2- ¹³ C ₂ ]AcCOA, defined as Φ _AcCit (isotope steady-state relationship):

The following equation uses ΦAcCit to correct the Q1/Q3 fragments of each Cit to eliminate the reverse ICDH flux and eliminate the effect of reverse flux:

195/70 _c = 195/70*196/70(1-Φ _AcCit )+195/69(1-Φ _AcCit ) (Equation 17)

194/70 _c = 194/70-195/69(1-Φ _AcCit ) (Equation 19)

194/69 _c = 194/69 - 195/69 (1 - Φ _AcCit ) + 194/68 (1 - Φ _AcCit ) (Equation 20)

193/69 _c = 193/69-194/68(1-Φ _AcCit ) (Equation 22)

193/68 _c = 193/68 - 194/68 (1 - Φ _AcCit ) + (193/68 - 193/69) (1 - Φ _AcCit ) (Equation 23)

(10) Mass isotope analysis distribution - calculation of isotopic enrichment of acetyl-CoA and oxaloacetate

If the enrichment of pyruvate and AcCOA is known, the relative contribution 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 the mitochondrial matrix pool of AcCOA and OAA. The present invention can solve this problem through mass isotope distribution analysis (MIDA).

For the reaction A+B→AB, if both substrates are partially enriched (FE _A* and FE _B* ), then MIDA will determine the enrichment of both precursors, even in the presence of dilution from external unlabeled products. The fractional enrichment (FE) of the substrate is defined as:

Where (*) indicates the presence of a measurable tag.

The reaction of (A+A ^* ) with (B+B ^* ) will produce AB+AB ^* +A ^* B+A ^* B ^* =1.

If there is unmarked contamination A'B', then AB+AB ^* +A ^* B+A ^* B ^* +A'B'=1.

The probability of generating a doubly labeled product (DA _*B* ) and a singly labeled substrate ( _SA*B and _SAB* ) is determined by the fractional enrichment of the formed products:

The following equations 29 and 30 describe the ratio of the single-labeled product to the double-labeled product:

Solving the above equations yields the equation for FE:

Cit is formed in the mitochondrial matrix by condensing AcCOA and OAA using CS. Mass spectrometry can evaluate single molecules, so D _A*B* , S _A*B and S _AB* can be determined from the deconvolution of the isotope family (see Deconvolution of citrate isotopes). The inventors found that there are several possible ways to calculate the partial enrichment of labeled AcCOA (FE _A* ) and labeled OAA (FE _B* ) in the mitochondrial matrix. In summary, the steady-state enrichment of OAA and AcCOA can be calculated by analyzing the enrichment of several citrate isotopomers, because any given citrate isotopomer is the result of the enrichment product of OAA and AcCOA, for example, [U- ¹³ C ₆ ]Cit is the product of [U- ¹³ C ₄ ]OAA and [1,2- ¹³ C ₂ ]AcCOA;

The calculation method of [1,2- ¹³ C ₂ ]AcCOA is as follows:

The calculation equation for ¹³ C-labeled OAA is as follows:

(11) Calculation of metabolic flux rate ratio based on isotope steady state

Steady-state isotope analysis can determine whether there is a significant net influx or exchange of unlabeled metabolites between consecutive or tandem metabolic reactions. One or more metabolic pathways may have enzymatic reactions that generate products, such as AcCOA formation primarily from PDH or β-oxidation of fatty acids or certain amino acids. If one of the pathways can be selectively labeled (e.g., pyruvate dehydrogenation or pyruvate carboxylation), the relative sources of carbon flowing into the pathway can be determined. Differential equations can be used to describe the rate of change of enrichment of substrate-labeled metabolites due to metabolic influx (influx rate minus efflux rate). For the generalized reaction:

Where A is the initial substrate that is converted to product B by enzyme _E1 , and then B is converted to C by enzyme _E2 , then the general equation is:

Under metabolic and isotopic stability conditions, the change in ¹³ C concentration over time is defined as zero. So the pathway _E1 to _E2 can be solved with respect to ( _Φ1→2 ) such that the relative contribution of the input to the output is equal to the enrichment of the product over its precursor.

If V _E1 is the only pathway that contributes to the production of B, V _E1 /V _E2 will be close to 1. However, this steady-state isotope analysis can only detect 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 values less than 1 indicate unlabeled input from another source. In simple terms, Φ _AB refers to the relative contribution of substrate A to the pathway that produces product B, that is, the metabolic rate ratio, while 1-Φ _AB represents the unlabeled input of this pathway. It should be noted that this analysis cannot distinguish between anaplerotic and exchange reactions, nor can it identify carbon losses from anaplerotic reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an experimental flow chart of the present invention, wherein the cells are labeled with isotopes and then subjected to liquid chromatography-mass spectrometry analysis, and finally the data are analyzed to obtain the precise metabolic flow of the isotopes;

FIG. 2 shows the serial nomenclature of carbon atoms in citric acid according to the present invention.

FIG3 is a fragment characterization of the parent ion and daughter ion of the deconvoluted isotopomers of citric acid in the present invention;

FIG4 is a schematic diagram of a 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 pathways flow out of acetyl-CoA;

FIG5 is a graph showing the results of measuring muscle metabolic sensitivity of rats under insulin resistance using glucose oxidation (left figure) and V _PDH /V _CS (right figure) as indicators in Example 2 of the present invention;

FIG6 is a schematic diagram of a metabolic pathway centered on oxaloacetate/malate in Example 3 of the present invention, in which both the pyruvate decarboxylase and citrate synthase pathways flow into oxaloacetate/malate, while the phosphoenolpyruvate carboxykinase and citrate synthase pathways flow out of oxaloacetate/malate;

FIG. 7 is a diagram showing changes in insulin secretion (A) and Φ _PO (B) of INS-1 under stimulation of different glucose concentrations in Example 3 of the present invention.

DETAILED DESCRIPTION

The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

Example 1

A method for accurately analyzing TCA cycle in pancreatic islet cells based on isotope tracing technology combined with high-resolution mass spectrometry, comprising the following steps:

(1) Preparation of culture medium:

The sugar-free medium was prepared by adding 0.2% BSA, 24 mM sodium bicarbonate, and 10 mM HEPES to DMEM medium (sigma-D5030), mixing with a magnetic stirrer, and adjusting the pH to 7.4;

The high-glucose medium (first medium) was prepared by adding 0.2% BSA, 24 mM sodium bicarbonate, 10 mM HEPES, and 20 mM glucose to DMEM medium (sigma-D5030), mixing with a magnetic stirrer, and adjusting the pH to 7.4;

The prepared culture medium was filtered with a 0.22 μM filter membrane, 0.1% penicillin-streptomycin mixture was added, and stored at 4°C;

The preparation method of ¹³ C-labeled high-glucose medium (second medium) is as follows: 0.2% BSA, 24 mM sodium bicarbonate, 10 mM HEPES, and 20 mM ¹³ C-labeled glucose are added to DMEM medium (sigma-D5030), mixed with a magnetic stirrer, and the pH is adjusted to 7.4;

The prepared culture medium was filtered with a 0.22 μM filter membrane, and a 0.1% penicillin-streptomycin mixture was added and stored at 4°C;

(2) Pancreatic islet cell culture:

When the islet cell density reached 80%, the islet cells were digested and seeded into a six-well plate with a complete culture medium (80% high glucose medium + 10% FBS) at a density of 5×10 ⁵ cells/well. After the islet cells adhered to the wall and the density reached 80%, they were starved for 2 h with a sugar-free culture medium. The islet cells were cultured with the first culture medium for 3 h to achieve cell metabolic homeostasis, and then the islet cells were labeled with a second culture medium containing the isotope ¹³ C for 3 h to achieve cell isotope homeostasis.

(3) Cell quenching:

Precool 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-shaped 96-well plates on ice. After the islet cell culture is completed, place the 96-well plate on ice, quickly aspirate the culture medium, add 2 mL of precooled (4°C) 5 mM HEPES to each well to wash the islet cells, then aspirate it, add 150 μL of ice-cold quenching buffer, scrape the islet cells on the culture medium with a scraper, and quickly transfer the islet cells and quenching buffer mixture to the 96-well plate on ice. Seal the 96-well plate with aluminum film, use a 20 g needle to poke a hole in each well, store it in a –80°C freezer overnight, and then freeze-dry;

(4) Liquid sample preparation:

Prepare samples before mass spectrometry analysis. Precool the centrifuge to 4°C, place the V-shaped 96-well plate on ice, resuspend the freeze-dried islet cell powder in precooled 50ul ultrapure water (containing 25μM taurine), and centrifuge the 96-well plate at 4000r/min for 5min. Transfer the supernatant to a new 96-well plate, repeat the above operation at least 3 times to ensure that there is no precipitate in the supernatant, transfer it to a sample bottle, and for quality control, create a mixed sample well by extracting 2.50μL from each well;

(5) Liquid chromatography-mass spectrometry analysis

A single standard was dissolved in 50% 1M HCl and 50% methanol to prepare a 5 mM stock solution. On the day of the experiment, ultrapure water containing 25 μM taurine (internal standard) was used to gradient dilute the mixed standard to 200/100/50/25/12.5/6.75/3.125/1.5625/0.78125 μM to draw a standard curve. After the analysis of 10 samples was completed, the mixed sample well was analyzed once. The cell sample extract cultured in the stable isotope ¹³ C labeled culture medium was analyzed by mass spectrometry in the mass spectrometry multiple reaction monitoring mode (MRM mode). The liquid phase mass spectrometry analysis of the target metabolites was carried out on the Waters Acquity UPLC system. The chromatographic conditions were as follows:

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

Mobile phase: Phase A: 0.1% formic acid in water, and Phase B: 0.1% formic acid in acetonitrile;

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: 35°C;

Sample cell temperature: 4°C;

Mobile phase flow rate: 0.4 mL/min;

Injection volume: 1 μL;

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

Capillary voltage in negative ion mode: 2.50 kV;

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

Desolventizing gas flow rate: 1000L/h;

Temperature: 550℃;

Cone gas flow rate: 150L/h;

Atomizer setting: 7.0Bar;

Data were collected by screening parent/daughter ions simultaneously in MRM mode, all of which were controlled by MassLynx software;

Table 1

(6) Background correction of liquid quality data

The peak area data were exported in MassLynx software, and the peak area of each ¹³ C-labeled sample was subtracted from the peak area of the normal ¹² C sample to obtain the background-corrected peak area of each sample.

(7) Natural abundance correction

In order to remove the influence of ¹³ C in nature, the measured ¹³ C is corrected for natural abundance to obtain the ¹³ C abundance added in the experiment. The natural abundance is adjusted by 1.1%. The corrected isotope matrix I' _{(Pm, Dn)} is to explain the presence of natural abundance carbon for each possible parent/daughter ion combination in the matrix I _{(Pm, Dn)} :

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))

p: total number of carbons in the parent ion;

d: total number of carbons in the product ion;

m: the number of ¹³ C in the parent ion;

n: the number of ¹³ C in the product ion;

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

k = 0.11 (natural abundance of ¹³ C in nature);

m-v≤p-d.;

(8) Deconvolution of citric acid

^13C glucose will produce ^13C pyruvate, which enters the TCA cycle through the pyruvate dehydrogenase (PDH) pathway and the pyruvate decarboxylase (PC) pathway. Citric acid (Cit) is a symmetrical molecule, but it contains a prochiral center, which can be distinguished by the stereochemical characteristics of the tricarboxylic acid cycle enzymes (as shown in Figure 2); acetyl coenzyme A (AcCOA) provides C4 and C5 of citric acid, and oxaloacetate (OAA) provides C1, 2, 3 and 6. If all Cit groups with ^13C labels at positions 4 and 5 (Cit _{a, d, h, f, i, j} , as shown in Figure 3) can be measured, the carbon atoms produced by the PDH pathway can be analyzed. Similarly, the carbon atoms produced by PC are also represented by the ^13C -labeled Cit group (Cit _{c, h} );

In the fragmentation mode, the C1, C6 or C5, C6 of Cit are cut off to obtain the daughter ions. Since these two carbon atoms contain information from the PC pathway and the PDH pathway, the metabolic flow information of the Cit family can be obtained by deconvolving all the daughter ions that have lost the two carbon atoms.

Cit without ¹³ C label is M, Cit with 1 to 6 ¹³ C labels are represented as [M+2], [M+3], [M+4], [M+5], [M+6], and the mother/daughter combination assignments of the single [M+2] to [M+6] isotope groups are as follows:

There are two ways to produce Cit[M+2] with two labels: one is Cit _a produced from the PDH pathway, and the other is Cit _a and Cit _b produced from the PDH pathway in the second round of TCA cycle. The fragments of Cit _a can produce equal amounts of 193/68 and 193/69 daughter ions, and the fragments of Cit _b can produce 3/4 of 193/68 daughter ions and 1/4 of 193/69 daughter ions. From this, we can derive the equation describing the isotopic composition of the parent/daughter combination:

Equations 1 and 2 can be solved to obtain:

Ci _a =3*193/69-193/68 (Equation 3)

Cit _b = 2*(193/68-193/69) (Equation 4)

Similarly, we can conclude that:

Cit _c =3*194/69-5*194/68-5*194/70 (Equation 5)

Cit _d = 4*194/68-4*194/69+12*194/70 (Equation 6)

Cit _e =2*194/68-2*194/69-6*194/70 (Equation 7)

Cit _f = 2*195/70+2*195/69 (Equation 8)

Cit _g =9*195/69-195/70 (Equation 9)

Cit _j = 197/71 (Equation 12);

(9) Correction of isocitrate dehydrogenase

The deconvolution process assumes that carbon flows directly from Cit to α-ketoglutarate (αKG). However, due to the presence of isocitrate dehydrogenase (ICDH), the reaction is reversible and αKG is also converted back to Cit. This reverse flux will affect the isotope labeling pattern of Cit. This reverse flux of ICDH will only affect Cit containing ¹³ C labeling at C6, while the labels of C4 and C5 are not affected. The correlation of this flux is proportional to the sum of all [4,5- ¹³ C]Cit families (∑Cit _a , Cit _f , Cit _i , Cit _h , Cit _d , Cit _j ) and [1,2- ¹³ C ₂ ]AcCOA, defined as Φ _AcCit (isotope steady-state relationship):

The following equation uses ΦAcCit to correct the Q1/Q3 fragments of each Cit to eliminate the reverse ICDH flux and eliminate the effect of reverse flux:

195/70 _c = 195/70*196/70(1-Φ _AcCit )+195/69(1-Φ _AcCit ) (Equation 17)

194/70 _c = 194/70-195/69(1-Φ _AcCit ) (Equation 19)

194/69 _c = 194/69 - 195/69 (1 - Φ _AcCit ) + 194/68 (1 - Φ _AcCit ) (Equation 20)

193/69 _c = 193/69-194/68(1-Φ _AcCit ) (Equation 22)

193/68 _c = 193/68 - 194/68 (1 - Φ _AcCit ) + (193/68 - 193/69) (1 - Φ _AcCit ) (Equation 23)

(10) Mass isotope analysis distribution - calculation of isotopic enrichment of acetyl-CoA and oxaloacetate

If the enrichment of pyruvate and AcCOA is known, the relative contribution 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 the mitochondrial matrix pool of AcCOA and OAA. The present invention can solve this problem through mass isotope distribution analysis (MIDA).

For the reaction A+B→AB, if both substrates are partially enriched (FE _A* and FE _B* ), then MIDA will determine the enrichment of both precursors, even in the presence of dilution from external unlabeled products. The fractional enrichment (FE) of the substrate is defined as:

Where (*) indicates the presence of a measurable tag.

The reaction of (A+A ^* ) with (B+B ^* ) will produce AB+AB ^* +A ^* B+A ^* B ^* =1.

If there is unmarked contamination A'B', then AB+AB ^* +A ^* B+A ^* B ^* +A'B'=1.

The probability of generating a doubly labeled product (DA _*B* ) and a singly labeled substrate ( _SA*B and _SAB* ) is determined by the fractional enrichment of the formed products:

The following equations 29 and 30 describe the ratio of the single-labeled product to the double-labeled product:

Solving the above equations yields the equation for FE:

Cit is formed in the mitochondrial matrix by condensing AcCOA and OAA using CS. Mass spectrometry can evaluate single molecules, so D _A*B* , S _A*B and S _AB* can be determined from the deconvolution of the isotope family (see Deconvolution of citrate isotopes). The inventors found that there are several possible ways to calculate the partial enrichment of labeled AcCOA (FE _A* ) and labeled OAA (FE _B* ) in the mitochondrial matrix. In summary, the steady-state enrichment of OAA and AcCOA can be calculated by analyzing the enrichment of several citrate isotopomers, because any given citrate isotopomer is the result of the enrichment product of OAA and AcCOA, for example, [U- ¹³ C ₆ ]Cit is the product of [U- ¹³ C ₄ ]OAA and [1,2- ¹³ C ₂ ]AcCOA;

The calculation method of [1,2- ¹³ C ₂ ]AcCOA is as follows:

The calculation equation for ¹³ C-labeled OAA is as follows:

(11) Calculation of metabolic flux rate ratio based on isotope steady state

Steady-state isotope analysis can determine whether there is a significant net influx or exchange of unlabeled metabolites between consecutive or tandem metabolic reactions. One or more metabolic pathways may have enzymatic reactions that generate products, such as AcCOA formation primarily from PDH or β-oxidation of fatty acids or certain amino acids. If one of the pathways can be selectively labeled (e.g., pyruvate dehydrogenation or pyruvate carboxylation), the relative sources of carbon flowing into the pathway can be determined. Differential equations can be used to describe the rate of change of enrichment of substrate-labeled metabolites due to metabolic influx (influx rate minus efflux rate). For the generalized reaction:

Where A is the initial substrate that is converted to product B by enzyme _E1 , and then B is converted to C by enzyme _E2 , then the general equation is:

Under metabolic and isotopic stability conditions, the change in ¹³ C concentration over time is defined as zero. So the pathway _E1 to _E2 can be solved with respect to ( _Φ1→2 ) such that the relative contribution of the input to the output is equal to the enrichment of the product over its precursor.

If V _E1 is the only pathway that contributes to the production of B, V _E1 /V _E2 will be close to 1. However, this steady-state isotope analysis can only detect 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 values less than 1 indicate unlabeled input from another source. In simple terms, Φ _AB refers to the relative contribution of substrate A to the pathway that produces product B, that is, the metabolic rate ratio, while 1-Φ _AB represents the unlabeled input of this pathway. It should be noted that this analysis cannot distinguish between anaplerotic and exchange reactions, nor can it identify carbon losses from anaplerotic reactions.

Example 2

The above-mentioned method of accurate metabolic flux analysis of TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry was used to explore the correlation between rat muscle metabolic substrate preference and metabolic rate ratio V _PDH /V _CS under insulin stimulation. The steps are as follows:

(1) Rat breeding

Male Sprague-Dawley rats (3 per cage) of 250 g were group-housed and divided into two groups (control group and insulin resistance group) and fed a normal diet or a high-fat diet for 3 consecutive weeks. All rats then underwent surgery under general isoflurane anesthesia, polyethylene catheters were placed in the common carotid artery (PE50 tubing) and jugular vein (PE90 tubing), and they were housed individually. The rats were fed their respective diets for 1 week before the studies were performed. All in vivo studies in rats were performed after a 16-h overnight fast. At the end of each study, the rats were euthanized by intravenous injection of pentobarbital.

(2) [U- ¹³ C ₆ ] glucose tracer enrichment experiment in vivo

In all studies, the isotopic tracer ([U- ¹³ C ₆ ]glucose) was infused through a catheter placed in the carotid artery 1 week earlier, and blood was obtained from a catheter in the jugular vein. All studies were initiated 1 hour after catheterization to minimize any effects of the rat's stress response on the assessed physiology.

To measure V _PDH /V _CS flux in the basal fasting state, rats were infused with [U- ¹³ C ₆ ]glucose (initial 3 mg/[Kg-min] for 5 min, followed by a continuous infusion rate of 1 mg/[Kg-min]). Rats were killed after a total infusion of 120 min, and skeletal muscles were snap-frozen in situ using metal clamps precooled in liquid nitrogen.

To measure V _PDH /V _CS flux under insulin stimulation, rats were given a 40 mU/Kg regular insulin bolus followed by an insulin infusion at a rate of 4.0 mU/(kg-min) while maintaining normoglycemia with a variable infusion of [U- ¹³ C ₆ ]glucose. Rats were sacrificed after a total of 120 min of infusion and skeletal muscle was snap frozen in situ using metal clamps precooled in liquid nitrogen.

(3) Liquid mass spectrometry sample preparation

100 mg of rat skeletal muscle sample was weighed and 0.1 mmol of taurine (internal standard) was added. The sample was homogenized in 500 ul of ice-cold methanol using a TissueLyser and filtered through a Nanosep filter.

(4) Liquid chromatography-mass spectrometry analysis

A single standard was dissolved in 50% 1M HCl and 50% methanol to prepare a 5 mM stock solution. On the day of the experiment, ultrapure water containing 0.1 mmol taurine (internal standard) was used to gradient dilute the mixed standard to 200/100/50/25/12.5/6.75/3.125/1.5625/0.78125 μM to draw a standard curve. After 10 samples were analyzed, the mixed sample well was analyzed once. The cell sample extract cultured in the stable isotope ¹³ C labeled culture medium was analyzed by mass spectrometry in the mass spectrometry multiple reaction monitoring mode (MRM mode). The liquid phase mass spectrometry analysis of the target metabolites was carried out on the Waters Acquity UPLC system. The chromatographic conditions were as follows:

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

Mobile phase: Phase A: 0.1% formic acid in water, and Phase B: 0.1% formic acid in acetonitrile;

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: 35°C;

Sample cell temperature: 4°C;

Mobile phase flow rate: 0.4 mL/min;

Injection volume: 1 μL;

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

Capillary voltage in negative ion mode: 2.50 kV;

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

Desolventizing gas flow rate: 1000L/h;

Temperature: 550℃;

Cone gas flow rate: 150L/h;

Atomizer setting: 7.0Bar;

Data were collected by screening parent/daughter ions simultaneously in MRM mode, all of which were controlled by MassLynx software;

Table 1

(5) HPLC data background correction

The peak area data were exported in MassLynx software, and the peak area of each ¹³ C-labeled sample was subtracted from the peak area of the normal ¹² C sample to obtain the background-corrected peak area of each sample.

(6) Natural abundance correction

In order to remove the influence of ¹³ C in nature, the measured ¹³ C is corrected for natural abundance to obtain the ¹³ C abundance added in the experiment. The natural abundance is adjusted by 1.1%. The corrected isotope matrix I' _{(Pm, Dn)} is to explain the presence of natural abundance carbon for each possible parent/daughter ion combination in the matrix I _{(Pm, Dn)} :

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))

p: total number of carbons in the parent ion;

d: total number of carbons in the product ion;

m: the number of ¹³ C in the parent ion;

n: the number of ¹³ C in the product ion;

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

k = 0.11 (natural abundance of ¹³ C in nature);

m-v≤p-d.;

(7) Calculation of metabolic flux ratio V _PDH /V _CS

PDH converts [U- ¹³ C ₃ ]pyruvate to [1,2- ¹³ C ₂ ]AcCOA, which can be further oxidized by citrate synthase (CS) (Figure 4). The temporal changes of [1,2- ¹³ C ₂ ]AcCOA and the mass balance relationship are described by the following equations 41 and 42. Since [U- ¹³ C ₃ ]PEP better represents the true enrichment of glycolytic precursors, we replaced [U- ¹³ C ₃ ]pyruvate with [U- ¹³ C ₃ ]PEP enrichment in all the following equations.

V _PDH +V _βOX =V _CS (Equation 42)

At steady state, Equation 1 can be further simplified and solved for V _PDH /V _CS to obtain the relative flux of PDH to CS (Φ _PAc ) (Equation 3). The enrichment in [1,2- ¹³ C ₂ ]AcCoA can be calculated as described in “Calculation of AcCoA enrichment”, while [U- ¹³ C ₃ ]PEP can be measured directly.

Although calculation of the true V _PDH /V _CS requires comparison of the ratio of [U- ^{13 C 3 ]PEP to [1,2- 13 C 2 ]AcCOA enriched intracellularly by the [U- 13 C 6 ]glucose tracer, these metabolites are rapidly degraded in vitro and thus difficult to measure. Under steady-state conditions, intracellular [U- 13} _{C 3} ^] _pyruvate ^is _assumed ^to _be in equilibrium with [U- ¹³ C ₃ ]alanine, and [1,2- ¹³ C ₂ ]AcCOA is assumed to be in equilibrium with [4,5- ¹³ C ₂ ]glutamate. Alanine and glutamate are more stable in vitro relative to their respective counterparts and are more suitable for reliable and consistent measurements of their intracellular enrichment. Therefore, V _PDH /V _CS flux was measured as the ratio of [4,5- ¹³ C ₂ ]glutamate to [U- ¹³ C ₃ ]alanine in skeletal muscle 2 h after infusion of [13C6]glucose.

(8) Analysis of the rate ratio V _PDH /V _CS

In the metabolic pathway, the flux ratio of V ₁ /V ₂ related to the change of metabolic pathway is calculated. This ratio can be used as an important indicator to measure the mechanism and influence of the conditions to be analyzed on the metabolic pathway. By changing the control conditions and observing the changes in the V ₁ /V ₂ ratio, it is possible to determine whether the conditions to be analyzed will affect the pathway, how they affect the pathway, and the specific changes caused. The metabolic rate ratio V _PDH /V _CS is a typical example, which reflects the proportion of glucose oxidation in mitochondria to the total mitochondrial material oxidation.

As shown in FIG5 , in order to verify whether the mitochondrial substrate preference changes in rats with high-fat-induced insulin resistance, the inventors injected [U- ¹³ C ₆ ] glucose into rats, and then detected the production of [4,5- ¹³ C ₂ ] glutamate and [U- ¹³ C ₃ ] alanine to characterize the metabolic flux of the PDH pathway (V _PDH ) and the metabolic flux of citric acid production (V _CS ), and the ratio of V _PDH /V _CS reflects the proportion of glucose oxidation in total mitochondrial oxidation. Whether normal or insulin-resistant rat skeletal muscle is stimulated by insulin, the V _PDH /V _CS ratio is significantly increased, although the increase ratio is reduced in insulin-resistant rats. This shows that even in rats with insulin resistance, the metabolic mode of its muscle tissue can still flexibly utilize glucose.

Example 3

The above-mentioned method of accurate metabolic flux analysis of TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry was used to explore the correlation between the changes in the target metabolic pathway and the metabolic rate ratio _VPC / _VCS . The steps are as follows:

(1) Preparation of culture medium:

The sugar-free medium was prepared by adding 0.2% BSA, 24 mM sodium bicarbonate, and 10 mM HEPES to DMEM medium (sigma-D5030), mixing with a magnetic stirrer, and adjusting the pH to 7.4;

The method for preparing the culture medium 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 pyruvic acid and 0.45 mM lactic acid were added to DMEM medium (sigma-D5030), and finally 2.5, 5, 7 or 9 mM glucose was added respectively, mixed with a magnetic stirrer, and the pH was adjusted to 7.4;

The prepared culture medium was filtered with a 0.22 μM filter membrane, 0.1% penicillin-streptomycin mixture was added, and stored at 4°C;

The method for preparing the culture medium containing different concentrations of ¹³ C-labeled 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 pyruvic acid and 0.45 mM lactic acid were added to DMEM culture medium (sigma-D5030), and finally 2.5, 5, 7 or 9 mM [U- ¹³ C ₆ ] glucose was added, mixed with a magnetic stirrer, and the pH was adjusted to 7.4;

The prepared culture medium was filtered with a 0.22 μM filter membrane, and a 0.1% penicillin-streptomycin mixture was added and stored at 4°C;

(2) Pancreatic islet cell (INS-1) culture:

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 adding the isotope label. They were then washed with DMEM medium without glucose and subsequently incubated for 3 hours with 2.5, 5, 7, and 9 mM [U- ¹³ C ₆ ] glucose to reach isotope stability.

(3) Cell quenching:

Precool 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-shaped 96-well plates on ice. After the INS-1 cell culture is completed, place the 96-well plate on ice, quickly aspirate the culture medium, add 2 mL of precooled (4°C) 5 mM HEPES to each well to wash the INS-1 cells, then aspirate it, add 150 μL of ice-cold quenching buffer, scrape the INS-1 cells on the culture medium with a scraper, and quickly transfer the INS-1 cells and quenching buffer mixture to the 96-well plate on ice. Seal the 96-well plate with aluminum film, use a 20 g needle to poke a hole in each well, store it in a –80°C freezer overnight, and then freeze-dry it;

(4) Liquid sample preparation:

Prepare samples before mass spectrometry analysis. Precool the centrifuge to 4°C, place the V-shaped 96-well plate on ice, resuspend the freeze-dried INS-1 cell powder in precooled 50ul ultrapure water (containing 25μM taurine), and centrifuge the 96-well plate at 4000r/min for 5min. Transfer the supernatant to a new 96-well plate, repeat the above operation at least 3 times to ensure that there is no precipitate in the supernatant, transfer it to a sample bottle, and for quality control, create a mixed sample well by extracting 2.50μL from each well;

(5) Liquid chromatography-mass spectrometry analysis

A single standard was dissolved in 50% 1M HCl and 50% methanol to prepare a 5 mM stock solution. On the day of the experiment, ultrapure water containing 25 μM taurine (internal standard) was used to gradient dilute the mixed standard to 200/100/50/25/12.5/6.75/3.125/1.5625/0.78125 μM to draw a standard curve. After 10 samples were analyzed, the mixed sample well was analyzed once. The cell sample extract cultured in the stable isotope ¹³ C labeled culture medium was analyzed by mass spectrometry in the mass spectrometry multiple reaction monitoring mode (MRM mode). The liquid phase mass spectrometry analysis of the target metabolites was carried out on the Waters Acquity UPLC system. The chromatographic conditions were as follows:

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

Mobile phase: Phase A: 0.1% formic acid in water, and Phase B: 0.1% formic acid in acetonitrile;

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: 35°C;

Sample cell temperature: 4°C;

Mobile phase flow rate: 0.4 mL/min;

Injection volume: 1 μL;

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

Capillary voltage in negative ion mode: 2.50 kV;

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

Desolventizing gas flow rate: 1000L/h;

Temperature: 550℃;

Cone gas flow rate: 150L/h;

Atomizer setting: 7.0Bar;

Data were collected by screening parent/daughter ions simultaneously in MRM mode, all of which were controlled by MassLynx software;

Table 1

(6) Background correction of liquid quality data

The peak area data were exported in MassLynx software, and the peak area of each ¹³ C-labeled sample was subtracted from the peak area of the normal ¹² C sample to obtain the background-corrected peak area of each sample.

(7) Natural abundance correction

In order to remove the influence of ¹³ C in nature, the measured ¹³ C is corrected for natural abundance to obtain the ¹³ C abundance added in the experiment. The natural abundance is adjusted by 1.1%. The corrected isotope matrix I' _{(Pm, Dn)} is to explain the presence of natural abundance carbon for each possible parent/daughter ion combination in the matrix I _{(Pm, Dn)} :

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))

p: total number of carbons in the parent ion;

d: total number of carbons in the product ion;

m: the number of ¹³ C in the parent ion;

n: the number of ¹³ C in the product ion;

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

k = 0.11 (natural abundance of ¹³ C in nature);

m-v≤p-d.;

(8) Deconvolution of citric acid

^13C glucose will produce ^13C pyruvate, which enters the TCA cycle through the pyruvate dehydrogenase (PDH) pathway and the pyruvate decarboxylase (PC) pathway. Citric acid (Cit) is a symmetrical molecule, but it contains a prochiral center, which can be distinguished by the stereochemical characteristics of the tricarboxylic acid cycle enzymes (as shown in Figure 2); acetyl coenzyme A (AcCOA) provides C4 and C5 of citric acid, and oxaloacetate (OAA) provides C1, 2, 3 and 6. If all Cit groups with ^13C labels at positions 4 and 5 (Cit _{a, d, h, f, i, j} , as shown in Figure 3) can be measured, the carbon atoms produced by the PDH pathway can be analyzed. Similarly, the carbon atoms produced by PC are also represented by the ^13C -labeled Cit group (Cit _{c, h} );

In the fragmentation mode, the C1, C6 or C5, C6 of Cit are cut off to obtain the daughter ions. Since these two carbon atoms contain information from the PC pathway and the PDH pathway, the metabolic flow information of the Cit family can be obtained by deconvolving all the daughter ions that have lost the two carbon atoms.

Cit without ¹³ C label is M, Cit with 1 to 6 ¹³ C labels are represented as [M+2], [M+3], [M+4], [M+5], [M+6], and the mother/daughter combination assignments of the single [M+2] to [M+6] isotope groups are as follows:

There are two ways to produce Cit[M+2] with two labels: one is Cit _a produced from the PDH pathway, and the other is the Cit _a and Cit _b produced from the PDH pathway in the second round of TCA cycle. The fragments of Cit _a can produce equal amounts of 193/68 and 193/69 daughter ions, and the fragments of Cit _b can produce 3/4 of 193/68 daughter ions and 1/4 of 193/69 daughter ions. From this, we can derive the equation describing the isotopic composition of the parent/daughter combination:

Equations 1 and 2 can be solved to obtain:

Ci _a =3*193/69-193/68 (Equation 3)

Cit _b = 2*(193/68-193/69) (Equation 4)

Similarly, we can conclude that:

Cit _c =3*194/69-5*194/68-5*194/70 (Equation 5)

Cit _d = 4*194/68-4*194/69+12*194/70 (Equation 6)

Cit _e =2*194/68-2*194/69-6*194/70 (Equation 7)

Cit _f = 2*195/70+2*195/69 (Equation 8)

Cit _g =9*195/69-195/70 (Equation 9)

Cit _j = 197/71 (Equation 12);

(9) Correction of isocitrate dehydrogenase

The deconvolution process assumes that carbon flows directly from Cit to α-ketoglutarate (αKG). However, due to the presence of isocitrate dehydrogenase (ICDH), the reaction is reversible and αKG is also converted back to Cit. This reverse flux will affect the isotope labeling pattern of Cit. This reverse flux of ICDH will only affect Cit containing ¹³ C labeling at C6, while the labels of C4 and C5 are not affected. The correlation of this flux is proportional to the sum of all [4,5- ¹³ C]Cit families (∑Cit _a , Cit _f , Cit _i , Cit _h , Cit _d , Cit _j ) and [1,2- ¹³ C ₂ ]AcCOA, defined as Φ _AcCit (isotope steady-state relationship):

The following equation uses ΦAcCit to correct the Q1/Q3 fragments of each Cit to eliminate the reverse ICDH flux and eliminate the effect of reverse flux:

195/70 _c = 195/70*196/70(1-Φ _AcCit )+195/69(1-Φ _AcCit ) (Equation 17)

194/70 _c = 194/70-195/69(1-Φ _AcCit ) (Equation 19)

194/69 _c = 194/69 - 195/69 (1 - Φ _AcCit ) + 194/68 (1 - Φ _AcCit ) (Equation 20)

193/69 _c = 193/69-194/68(1-Φ _AcCit ) (Equation 22)

193/68 _c = 193/68 - 194/68 (1 - Φ _AcCit ) + (193/68 - 193/69) (1 - Φ _AcCit ) (Equation 23)

(10) Mass isotope analysis distribution - calculation of isotopic enrichment of acetyl-CoA and oxaloacetate

If the enrichment of pyruvate and AcCOA is known, the relative contribution 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 the mitochondrial matrix pool of AcCOA and OAA. The present invention can solve this problem through mass isotope distribution analysis (MIDA).

For the reaction A+B→AB, if both substrates are partially enriched (FE _A* and FE _B* ), then MIDA will determine the enrichment of both precursors, even in the presence of dilution from external unlabeled products. The fractional enrichment (FE) of the substrate is defined as:

Where (*) indicates the presence of a measurable tag.

The reaction of (A+A ^* ) with (B+B ^* ) will produce AB+AB ^* +A ^* B+A ^* B ^* =1.

If there is unmarked contamination A'B', then AB+AB ^* +A ^* B+A ^* B ^* +A'B'=1.

The probability of generating a doubly labeled product (DA _*B* ) and a singly labeled substrate ( _SA*B and _SAB* ) is determined by the fractional enrichment of the formed products:

The following equations 29 and 30 describe the ratio of the single-labeled product to the double-labeled product:

Solving the above equations yields the equation for FE:

Cit is formed in the mitochondrial matrix by condensing AcCOA and OAA using CS. Mass spectrometry can evaluate single molecules, so D _A*B* , S _A*B and S _AB* can be determined from the deconvolution of the isotope family (see Deconvolution of citrate isotopes). The inventors found that there are several possible ways to calculate the partial enrichment of labeled AcCOA (FE _A* ) and labeled OAA (FE _B* ) in the mitochondrial matrix. In summary, the steady-state enrichment of OAA and AcCOA can be calculated by analyzing the enrichment of several citrate isotopomers, because any given citrate isotopomer is the result of the enrichment product of OAA and AcCOA, for example, [U- ¹³ C ₆ ]Cit is the product of [U- ¹³ C ₄ ]OAA and [1,2- ¹³ C ₂ ]AcCOA;

The calculation method of [1,2- ¹³ C ₂ ]AcCOA is as follows:

The calculation equation for ¹³ C-labeled OAA is as follows:

(11) Calculation of metabolic flux rate ratio based on isotope steady state

Steady-state isotope analysis can determine whether there is a significant net influx or exchange of unlabeled metabolites between consecutive or tandem metabolic reactions. One or more metabolic pathways may have enzymatic reactions that generate products, such as AcCOA formation primarily from PDH or β-oxidation of fatty acids or certain amino acids. If one of the pathways can be selectively labeled (e.g., pyruvate dehydrogenation or pyruvate carboxylation), the relative sources of carbon flowing into the pathway can be determined. Differential equations can be used to describe the rate of change of enrichment of substrate-labeled metabolites due to metabolic influx (influx rate minus efflux rate). For the generalized reaction:

Where A is the initial substrate that is converted to product B by enzyme _E1 , and then B is converted to C by enzyme _E2 , then the general equation is:

Under metabolic and isotopic stability conditions, the change in ¹³ C concentration over time is defined as zero. So the pathway _E1 to _E2 can be solved with respect to ( _Φ1→2 ) such that the relative contribution of the input to the output is equal to the enrichment of the product over its precursor.

If V _E1 is the only pathway that contributes to the production of B, then V _E1 /V _E2 will be close to 1. However, this steady-state isotope analysis can only detect 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 values less than 1 indicate unlabeled input from another source. In simple terms, Φ _AB refers to the relative contribution of substrate A to the pathway that produces product B, that is, the metabolic rate ratio, while 1-Φ _AB represents the unlabeled input of this pathway. It should be noted that this analysis cannot distinguish between anaplerotic and exchange reactions, nor can it identify carbon losses from anaplerotic reactions.

(12) Calculation of metabolic flux ratio V _PC /V _CS

Pyruvate decarboxylase (PC) is a mitochondrial enzyme that converts pyruvate to oxaloacetate (OAA) (Figure 6). The relative rates of synthesis of [(1,2,3)(2,3,4)- ¹³ C ₃ ]OAA from [U- ¹³ C ₃ ]pyruvate can be determined similarly to the description of PDH. In the case of metabolic homeostasis, OAA and malate can be considered as a proportional exchange due to the rapid exchange relative to TCA cycle flux. Therefore, both phosphoenolpyruvate carboxylase (PEPCK) and malic enzyme (ME) must be considered as reactions that consume [(1,2,3)(2,3,4)- ¹³ C ₃ ]OAA. The time course of [(1,2,3)(2,3,4)- ¹³ C ₃ ]OAA and the mass balance relationship are described by Equations 4 and 5, respectively.

V _PC +V _CS =V _CS +V _PEPCK +V _ME →V _PC =V _PEPCK +V _ME (Equation 44)

Under steady state conditions, Equations 4 and 5 can be further simplified and solved for V _PC /V _CS to obtain the relative flux from PC to CS (Φ _PO ) (Equations 46 and 47).

(13) Determination of insulin secretion in INS-1 cells The insulin concentration of medium extracts was measured using a rat high range insulin ELISA kit (ALPCO) according to the manufacturer's instructions. All medium extracts were normalized to cellular protein concentration using a Solebro-BCA protein assay kit.

(14) Relationship between rate ratio V _PC /V _CS and insulin secretion in INS-1 cells

Through experimental verification, the inventors found that the metabolic flux rate ratio V _PC /V _CS is closely related to glucose concentration and insulin secretion, and can be used in characterizing related cellular glucose metabolism and insulin secretion.

As a model system, pancreatic β cells link glucose-dependent changes in metabolism to the functional output of insulin secretion. The clonal insulinoma cell line INS-1 has become a model cell for many published experiments, and the amount of insulin released is linearly correlated within the physiological glucose concentration range (2.5-9mM) (as shown in Figure 7A). The inventors attempted to link changes in the metabolic flux rate ratio in metabolomics analysis to insulin secretion. Among them, during mitochondrial metabolic homeostasis, only changes in glucose concentration change proportionally with the relative flux (Φ _PO ) from PC to CS (as shown in Figure 7B). This shows that the metabolic flux rate ratio V _PC /V _CS can characterize the relationship between insulin secretion and glucose concentration, and has potential applications in characterizing cellular glucose metabolism and insulin secretion.

Claims (10)

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: cell culture: after starving the cells for 1-4 h, culturing the cells for 1-6 h by using a first culture medium to reach the metabolic steady state of the cells, and then using the cells with isotopes ¹³ Marking cells for 1-6 h by using a second culture medium of C to reach the isotope steady state of the cells;

s2: preparing a liquid sample: collecting cells by using 50-200 mu L of cell quencher and freeze-drying, then re-dissolving the freeze-dried cells by using 20-100 mu L/hole of complex solvent, and centrifuging to obtain supernatant fluid to prepare a liquid sample;

s3: carrying out liquid quality analysis by adopting a liquid quality analyzer;

s4: liquid analysis data processing;

s41: natural abundance correction;

s42: deconvolution of citric acid;

s43: correction of isocitrate dehydrogenase;

s44: measuring the contents of acetyl coenzyme A and oxaloacetic acid by mass isotope distribution analysis;

s45: metabolic flux rate ratio calculation based on isotope steady state.

2. The method for analyzing TCA cycle based on isotope tracing technology and high resolution mass spectrometry according to claim 1, wherein the preparation method of the first culture medium in S1 is that 0.1-2.0% BSA, 12-30 mM sodium bicarbonate, 5-15 mM HEPES and 0-25 mM sugar substrate are added into DMEM culture medium, uniformly mixed by a magnetic stirrer, the pH is adjusted to 6.8-7.8, filtered by a 0.22 mu M filter membrane, and 0.1% of a mixture of green streptomycin is added and stored at 4 ℃; the preparation method of the second culture medium comprises the steps of adding 0.1-2% BSA, 12-30 mM sodium bicarbonate, 5-15 mM HEPES and 0-25 mM into a DMEM culture medium ¹³ Mixing the substrate marked by C with a magnetic stirrer, adjusting the pH to 6.8-7.8, filtering with a 0.22 mu M filter membrane, adding 0.1% of a mixture of the green streptomycin, and preserving at 4 ℃.

3. The method for analyzing the TCA cycle by combining the isotope labeling technique with the high-resolution mass spectrometry according to claim 2, wherein if the cells are hepatocytes, the glycogenic substrate is 1-20 mM lactic acid and 1-20 mM pyruvic acid, and if the cells are islet cells, the glycogenic substrate is 1-20 mM glucose and 1-20 mM glutamine, and the pH is adjusted to 6.8-7.8.

4. The method for analyzing the TCA cycle by combining the isotope labeling technique with the high-resolution mass spectrometry according to claim 1, wherein the cell quencher 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 mu M ethylenediamine tetraacetic acid; the complex solvent is 2-100 mu M taurine aqueous solution.

5. The method for analyzing TCA cycle based on isotope labeling technique combined with high-resolution mass spectrometry according to claim 1, wherein the liquid chromatograph in S3 is Waters Acquity UPLC system, (1) chromatographic conditions are:

chromatographic column: ACQUITY UPLC@HSS T3 chromatographic column, 2.1mm×100mm,1.8 μm;

mobile phase: phase A: 0.1% formic acid in water, and phase B: 0.1% acetonitrile formate solution;

the liquid phase gradient procedure was: 0.0 to 0.5min,99 percent of A;0.5 to 4.0min,99 to 95 percent of A;4.01 to 6.0min,99 percent of A;

column temperature: 20-55 ℃;

sample cell temperature: 4-10 ℃;

mobile phase flow rate: 0.2-0.5 mL/min;

sample injection amount: 1 μl;

(2) Mass spectrometry was performed using mass spectrometers Xevo TQ-S in electrospray ionization source anion detection mode, the source parameters of the mass spectrometry method were as follows:

Capillary voltage: 0.2-3.2 kV;

flow rate of desolventizing gas: 800-1200L/h;

temperature: 450-600 ℃;

taper hole gas flow rate: 100-250L/h;

atomizer setting: 5 to 10Bar.

6. The method for analyzing the TCA cycle based on the accurate metabolic flux of the isotope labeling technique combined with the high-resolution mass spectrum according to claim 1, wherein the natural abundance correction in S41 is performed by correcting the measured abundance ¹³ C correction of natural abundance to remove from nature ¹³ Influence of C, thereby obtaining an additive in the experiment ¹³ C abundance, 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))

and p: total number of carbons in the parent ion;

d: total number of carbons in the daughter ion;

m: in parent ion ¹³ Number of C;

n: in the ion of the son ¹³ Number of C;

i: peak areas corresponding to parent ion P from 0 to P and child ion D from 0 to D;

k=0.11, in nature ¹³ Natural abundance of C;

m-v≤p-d。

7. the method for analyzing TCA cycle by combining accurate metabolic flux with high-resolution mass spectrometry according to claim 1, wherein the sub-ion fragment information in the processing of the liquid analysis data in S42 is obtained by cutting C1, C6 or C5, C6 of Cit from parent ion to obtain sub-ion, and the two carbon atoms contain information from pyruvate decarboxylase pathway and pyruvate dehydrogenase pathway, and the citric acid family is obtained by deconvolution analysis of all sub-ion fragments: cit (Cit) _a 、Cit _b 、Cit _c 、Cit _d 、Cit _e 、Cit _f 、Cit _g 、Cit _h 、Cit _i 、Cit _j The metabolic flux information of (2) is calculated 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。

8. an isotope-based representation in accordance with claim 1A method for analyzing TCA cycle by combining the trace technique with high-resolution mass spectrometry (high-resolution mass spectrometry) with accurate metabolic flow, which is characterized in that the correction of isocitrate dehydrogenase in S43 is carried out by [4,5 ] ¹³ C ₂ ]Citric acid and [1,2 ] ¹³ C ₂ ]The ratio of acetyl-CoA corrects the marked carbon reflux phenomenon caused by the reverse flow of the isocitrate dehydrogenase reaction, so that the deconvolution result 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 )。

9. the method for analyzing TCA cycle by combining accurate metabolic flow based on isotope tracing technology with high resolution mass spectrometry according to claim 1, wherein the content of acetyl coa and oxaloacetate is determined by mass isotope analysis of citric acid in S44, and the calculation formula is as follows:

[1，2- ¹³ C ₂ ]the AcCOA calculation method is as follows:

¹³ the equation for the C-labeled OAA is calculated as follows:

10. a parity-based system as claimed in claim 1A method for analyzing TCA cycle by means of the precision metabolic flow analysis of the plain tracer technique in combination with high resolution mass spectrometry, characterized in that the metabolic flow rate ratio of the isotope steady state in S45 is calculated as determining by means of steady state isotope analysis whether there is a net entry, back-filling or exchange of unlabeled metabolites between consecutive or tandem metabolic reactions and calculating the rate ratio between metabolic pathways by means of differential equations:

To describe the rate of change of enrichment of the substrate-tagged metabolite due to metabolic changes. />

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