CN115950986A - Method for quantitatively analyzing central carbon metabolic intermediates - Google Patents
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Abstract
The invention discloses a method for quantitatively analyzing a central carbon metabolic intermediate, which comprises the following steps: respectively reacting light-weight 2-DMBA with the central carbon metabolic intermediate mixed standard substance with different concentrations to obtain a plurality of analytes with different concentrations of standard substances; at the same time, will re-mark d 5 Reacting 2-DMBA with the central carbon metabolic intermediate mixed standard to obtain a derivative of an intermediate as an internal standard; then performing liquid chromatography-mass spectrometry to obtain different chromatographic peaks and obtaining a linear regression equation through fitting; reacting light-weight 2-DMBA with a sample to be analyzed, performing liquid chromatography-mass spectrometry after the reaction, and simultaneously adding an internal standard to obtain the peak surface of a target detection object marked by the 2-DMBASum of products d 5 -ratio of peak areas of the 2-DMBA internal standard label; and substituting the ratio into the linear regression equation to calculate the concentration of the target detection object in the sample.
Description
Technical Field
The invention relates to the technical field of analysis, in particular to a method for quantitatively analyzing a central carbon metabolic intermediate.
Background
The Central carbon metabolism (Central carbon metabolism) pathway is one of the most important metabolic pathways present in all living bodies. The central carbon metabolism is not only an energy metabolic pathway of a living body to supply energy to the living body, but also provides precursor substances for biosynthesis of other metabolites. The occurrence and development of most diseases of a living body are closely related to central carbon metabolic pathways, such as cancers, cardiovascular diseases and the like. In view of its important role in all living bodies, it is of great importance to study the expression of metabolites associated therewith. The liquid chromatography-mass spectrometry (LC-MS) has the advantages of wide application range, high selectivity and high sensitivity, so that the LC-MS is widely applied to detection of endogenous small molecule metabolites. However, the central carbon metabolism intermediates have various structures and different physicochemical properties, so that the central carbon metabolism intermediates are difficult to be well separated and detected in the process of one-time LC-MS operation, and the responses on an electrospray ionization (ESI) mass spectrum are interfered and low-abundance intermediates are difficult to be detected due to the complex sample matrix.
Although hydrophilic interaction chromatography, ion pair chromatography and anion exchange chromatography can be used for separating partial phosphate sugar and carboxylic acid metabolic intermediates, the analysis effect on isomers of the central carbon metabolic intermediate is limited, and the mass spectrum response of an analyte cannot be enhanced; the metabolic intermediates analyzed by the current central carbon metabolic intermediate labeling reagent have limited types, limited detection coverage and limited analysis accuracy.
Therefore, it is necessary to develop a method for quantitatively analyzing the central carbon metabolic intermediate with wide coverage and high accuracy.
Disclosure of Invention
The invention aims to provide a method for quantitatively analyzing a central carbon metabolic intermediate, namely a stable isotope aromatic diazo reagent 2-DMBA/d 5 the-2-DMBA is used as a derivatization reagent to develop a simultaneous derivatization technology of the central carbon metabolic intermediate, and an accurate quantitative analysis method of the endogenous central carbon metabolic intermediate with high sensitivity, high selectivity and high coverage is established by combining LC-MS.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for quantitatively analyzing a central carbon metabolism intermediate, which comprises the following steps:
respectively reacting light-weight standard 2-DMBA with the central carbon metabolic intermediate mixed standard substance with different concentrations to obtain a plurality of analytes with different concentrations of standard substances; at the same time, will re-mark d 5 Reacting 2-DMBA with the central carbon metabolic intermediate mixed standard to obtain a derivative of the intermediate as an internal standard;
performing liquid chromatography-mass spectrometry on a plurality of analytes with different concentrations of standard substances and the internal standard substance to obtain different chromatographic peaks and obtaining a linear regression equation through fitting;
reacting light-weight 2-DMBA with a sample to be analyzed, performing liquid chromatography-mass spectrometry after the reaction, and simultaneously adding an internal standard to perform liquid chromatography-mass spectrometry to obtain the peak area and d of a target detection object marked by the 2-DMBA 5 -ratio of peak areas of the 2-DMBA internal standard labeling;
and substituting the ratio into the linear regression equation to calculate the concentration of the target detection object in the sample.
Further, in the liquid chromatography-mass spectrometry, the chromatographic separation conditions are as follows: the column used for the liquid phase separation was BEH C18 (2.1X 100mm,1.7 μm, waters.). The column temperature was 50 ℃ and the flow rate was 0.3mL/min. Mobile phases a and B were 2mM ammonium bicarbonate solution and acetonitrile, respectively. The mobile phase gradient is: 0-2 minutes, 5% B,2-17.5 minutes, 5% -25% B,17.5-21 minutes, 25% -90% B,21-24 minutes, 90% B,24-25 minutes, 90% -5%,25-30 minutes, 5%B.
Further, the step of reacting the light-labeled 2-DMBA with the mixed standard substance of the central carbon metabolic intermediate at different concentrations respectively to obtain a plurality of analytes with different concentrations of the standard substance comprises:
the mixed standard of central carbon metabolic intermediates is formulated into solutions of varying concentrations, including concentrations of 0,0.01,0.025,0.05,0.1,0.25,0.5,1,2.5,5,10,25,50,100,250,500ng/mL, and then labeled with 2-DMBA to obtain a plurality of analytes with standards of varying concentrations.
Further, the d to be relabeled 5 Reacting the (E) -2-DMBA with a mixed standard substance of the central carbon metabolic intermediate of 1ng/mL to obtain a derivative of the intermediate, wherein the derivative is used asInternal standard.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
the invention provides a method for quantitatively analyzing a central carbon metabolic intermediate, which is to mix 2-DMBA/d 5 The 2-DMBA is a pair of aromatic diazo reagents which are used for simultaneously labeling metabolic intermediates including phosphate sugar, carboxylic acid and coenzyme A in central carbon metabolism, and the mass spectrum response of the structure is better, and the detection sensitivity of the metabolic intermediates can be improved after the central carbon metabolic intermediates are labeled; the hydrophobicity is strong, the chromatographic retention can be enhanced, and the separation of reversed phase liquid chromatography is facilitated; and the introduction of the stable isotope label can provide an internal standard for a target analyte and improve the quantitative accuracy.
(1) A pair of stable isotope aromatic diazo reagents 2-DMBA/d 5 2-DMBA as a labeling reagent, reacting light-labeled 2-DMBA with a central carbon metabolic intermediate in a sample, and heavy-labeled d 5 -reacting 2-DMBA with a standard to obtain a derivative of a central carbon metabolic intermediate;
(2) And (3) mixing the derivatives obtained by the heavy and light marks, and then carrying out accurate quantitative analysis on the central carbon metabolic intermediate by adopting a liquid chromatography-mass spectrometry combination method, wherein the derivatives marked by the heavy marks are used as internal marks.
(3) The chemical labeling strategy for analyzing the endogenous central carbon metabolic intermediate can effectively improve the retention of a target object on a reversed phase chromatogram and can realize good separation of multiple pairs of isomers.
(4) The chemical labeling strategy for analyzing the endogenous central carbon metabolic intermediates realizes high coverage analysis, and can obtain accurate content information of various metabolic intermediates (including phosphoglucose, carboxylic acid and coenzyme A) in one sample injection.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a chemical structural formula of 22 targeted central carbon metabolic intermediates;
FIG. 2 is d 0 /d 5 -an equation for the structure of the 2-DMBA reagent reacted with the phosphate and carboxylic acid group labels;
FIG. 3 shows 5 representative structural intermediates labeled with 2-DMBA: a secondary mass spectrum of 6-phosphogluconic acid (a), acetyl coenzyme A (b), pyruvic acid (c), fumaric acid (d) and aconitic acid (e);
FIG. 4 shows the labeling efficiency of 2-DMBA for labeling central carbon metabolic intermediates;
FIG. 5 is a chromatogram of the separation of 2-DMBA labeled products for 22 analytes of interest. 1,3-phosphoglycerate; 2,2-phosphoglycerate; 3,6-phosphogluconic acid; 4, phosphoenolpyruvate; 5, glucose-6-phosphate; 6, heptose-7-phosphate; 7, fructose-6-phosphate; 8, ribose-5-phosphate; 9, xylulose-5-phosphate; 10, ribulose-5-phosphate; 11, erythrose-4-phosphate; 12, dihydroxyacetone phosphate; 13, acetyl-coenzyme a;14, pyruvic acid; 15, malic acid; 16, succinic acid; 17, fumaric acid; 18, α -ketoglutaric acid; 19, succinyl-coa; 20, citric acid + isocitric acid; 21, aconitic acid.
Detailed Description
The present invention will be specifically explained below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.
In order to solve the technical problems, the embodiments of the present invention search as follows:
2-DMBA/d based on a pair of stable isotope aromatic diazo reagents 5 Application of (E) -2-DMBA in LC-MS (liquid chromatography-mass spectrometry) analysis of phosphate compounds by combining diazo reagent-DMBA/d 5 Reaction of 2-DMBA with carboxylic acid groups and with acid groups, the principle of reaction, we speculate that 2-DMBA/d 5 2-DMBA such a pair of aryl diazonium reagents can be used for the simultaneous labeling of metabolic intermediates including phosphate sugars, carboxylic acids and coenzyme A in central carbon metabolism.
(1) First, we searched for a pair of stable isotope aryl diazonium reagents d 0 /d 5 -2- (diazomethyl) -N-methyl-N-phenylbenzamide (2-DMBA/d) 5 -2-DMBA) as a labeling reagent capable of chemically labeling the central carbon metabolic intermediate
The specific structure of 22 central carbon metabolism intermediates of our target relates to phosphoglucose, coenzyme A and carboxylic acid metabolites, and is shown in figure 1. We identify five central carbon metabolic intermediates with different representative structures: the method is characterized in that acetyl coenzyme A (coenzymes A), 6-phosphogluconate (simultaneously containing carboxyl and phosphate groups), pyruvic acid (monocarboxylic acid), fumaric acid (dicarboxylic acid) and aconitic acid (tricarboxylic acid) are used as models, and 2-DMBA is subjected to derivatization reaction with the models respectively, and experimental results show that central carbon metabolic intermediates with different structures can be effectively marked by 2-DMBA reagents. From the secondary mass spectra of five derivatives obtained by 2-DMBA labeling (FIG. 3), it can be seen that different derivatives can generate characteristic secondary fragments to assist in qualitative and quantitative analysis. In addition, we examined the conversion of this reaction in negative ion mode and found that the conversion was greater than 80% for all target products (fig. 4).
(2) Second, whether the labeled product could be separated by chromatography was analyzed
Chromatographic separation conditions: the column used for the liquid phase separation was BEH C18 (2.1X 100mm,1.7 μm, waters.). The column temperature was 50 ℃ and the flow rate was 0.3mL/min. Mobile phases a and B were 2mM ammonium bicarbonate solution and acetonitrile, respectively. The mobile phase gradient is: 0-2 minutes, 5% B,2-17.5 minutes, 5% -25% B,17.5-21 minutes, 25% -90% B,21-24 minutes, 90% B,24-25 minutes, 90% -5%,25-30 minutes, 5% B. The injection volume was 10. Mu.L.
Isomer separation results: the presence of multiple sets of isomers in the target analyte includes: (ii) (a) glucose-6-phosphate and fructose-6-phosphate; (b) 2-phosphoglycerate and 3-phosphoglycerate (c) ribose-5-phosphate, ribulose-5-phosphate nucleus, xylulose-5-phosphate; (d) citric acid and isocitric acid. These strongly hydrophilic acidic compounds were difficult to retain and separate on the C18 column before derivatization, and the derivatized products were retained on the C18 column after derivatization due to the increased hydrophobicity, and these central carbon metabolic intermediates were separated except for citric acid and isocitric acid (fig. 5). We optimized the performance parameters when detecting target analytes using the multiple reaction detection (MRM) mode under optimal chromatographic and mass spectral conditions (table 1).
TABLE 1.22 optimal MRM parameters for labeled products of central carbon metabolism intermediates
The above results show that a pair of stable isotope aromatic diazo reagents d 0 /d 5 -2- (diazomethyl) -N-methyl-N-phenylbenzamide (2-DMBA/d) 5 -2-DMBA) as labeling agent.
A method for quantitatively analyzing a central carbon metabolism intermediate according to the present application will be described in detail with reference to examples, comparative examples and experimental data. Diazo reagent d of the examples of the present invention 0 /d 5 -2- (diazomethyl) -N-methyl-N-phenylbenzamide (2-DMBA/d) 5 -2-DMBA) for laboratory synthesis, d 0 /d 5 The equation for the structure of the-2-DMBA reagent reacted with the phosphate and carboxylic acid group labels is shown in FIG. 2.
Example 1, 2-DMBA/d 5 -2-DMBA chemical isotope labeling strategy for quantitative analysis of target central carbon metabolism intermediates in HEK-293T cells
Synthesis of 2-DMBA: (1) First 2-carboxybenzaldehyde (225 mg), carbodiimide hydrochloride (EDCI, 580 mg) and 1-hydroxybenzotriazole (HOBt, 500 mg) were added sequentially to a 50-mL round-bottomed flask followed by 15mL Dichloromethane (DCM). After stirring for 30min, N-methylaniline (107 mg) was added and stirred at room temperature for 12h to give a crude product of 2-formyl-N-methyl-N-phenylbenzamide (2-FMBA). (2) The obtained crude product 2-FMBA was isolated and purified by silica gel column chromatography with hexane/ethyl acetate (5, 1,v/v) as an eluent. Collecting effluent liquid by using a conical flask, absorbing fractions by using a glass sample application capillary, applying the fractions to a silica gel plate to monitor whether ultraviolet absorption exists or not under the irradiation of an ultraviolet lamp with the wavelength of 254nm, starting to collect the fractions (2-FMBA) by using a test tube after the ultraviolet absorption occurs, observing the ultraviolet absorption condition through the point plate (the same as the above) at any time in the collection process, and stopping the collection when the absorption becomes very weak. Fractions collected from the tubes were pooled after further confirmation by mass spectrometry. The resulting pure 2-FMBA was spin dried (in vacuo) on a rotary evaporator in a water bath at 33 deg.C to yield the purified product of 2-FMBA as a pale beige clear oil. (3) Dissolve purified 2-FMBA in 10mL ethanol to give a nearly colorless clear solution (in a 50-mL round bottom flask), add N 2 H 4 ·H 2 O (250 mg). The mixture was magnetically stirred at room temperature for 8 hours, then rotary evaporated to remove ethanol. (4) To a rotary dry round bottom flask was added 8mL DCM and MnO 2 (435 mg) and stirred at room temperature for 4h. The sand-core funnel was filtered to remove solids and the final product, 2-DMBA solution, was collected in a 25-mL round bottom flask (weighed). The synthesized 2-DMBA solution was spin-dried in a water bath at 33 deg.C to give an oily product (orange), weighed, and the weight of the resulting product was calculated by subtraction. The 2-DMBA product is dissolved in DMSO to prepare a 10mg/mL stock solution, and the stock solution is subpackaged into 1.5-mL centrifuge tubes and stored in a refrigerator at the temperature of-20 ℃. The resulting solution was an orange solution. d 5 Synthesis of-2-DMBA using d 5 -aniline as raw material, by methylation to obtain d 5 N-methylaniline, the subsequent synthesis procedure being identical to that of 2-DMBA. In other embodiments, diazonium reagent d 0 /d 5 -2-(diazomethyl) -N-methyl-N-phenylbenzamide (2-DMBA/d) 5 -2-DMBA) can also be synthesized by other methods, and the structural formula is shown in figure 1.
Sample preparation: HEK-293T cells were harvested, washed 3 times with extraction solution, and then cold methanol/water (80% MeOH,20% H) was added 2 O, v/v) cell ultrasonication extraction in ice water bath. Centrifuging at high speed (14000g, 10 min), collecting supernatant, and storing. Stored at-80 ℃ for further use.
The detection steps of the HEK-293T cells are as follows: mu.L of borate buffer (50mM, pH 7) and 5. Mu.L of 2-DMBA were added to 10. Mu.L of the sample, and reacted at 30 ℃ for 1 hour after vortexing. After completion of the reaction, 10. Mu.L of internal standard was added and the whole was transferred to a 1.5 mL-vial. UHPLC-ESI-MS/MS sample injection analysis.
The analysis method of the central carbon metabolism intermediate in the HEK-293T cell sample comprises the following steps:
(1) Linear regression equation and correlation coefficient establishment of target detection object, detection limit and quantitative limit investigation
We prepared a mixture of 22 central carbon metabolic intermediates as standard solutions of different concentrations (0, 0.01,0.025,0.05,0.1,0.25,0.5,1,2.5,5,10,25,50,100,250, 500ng/mL) using 2-DMBA label, and added d separately 5 -2-DMBA-labeled central carbon metabolic intermediate cocktail standard (1 ng/mL) as an internal standard to assist in accurate quantitation to reduce co-effluent interference. After the analysis of RPLC-MRM-MS, the concentration of the corresponding central carbon metabolic intermediate IS subjected to linear regression treatment by using the ratio (analyte/IS) of the peak area of the standard substance of the central carbon 6 use metabolic intermediate marked by 2-DMBA to the peak area of the internal standard. The detection limits LODs and quantification limits LOQs were calculated at triple and ten signal-to-noise ratios, respectively (table 2).
TABLE 2.22 Linear Range of labeled products, LODs and LOQs for central carbon metabolism intermediates
(2) Intra-day, inter-day precision and accuracy:
recovery and day to day precision of all analytes were determined using matrix spiking. Wherein, the standard substance with different concentrations is respectively added into the substrate to obtain the standard substance with different concentrations, the precision in the day is examined by 5 different experiments in the same day, and the RSD of the standard substance with low, medium and high concentrations for 3 consecutive days is measured as the precision in the day. The accuracy of the method is measured by the addition standard recovery rate of the matrix samples with low, medium and high concentrations.
TABLE 3.22 Intra-day, inter-day precision and accuracy of the labeled products of the central carbon metabolism intermediates
And (3) detection results: the method has good linearity and linear coefficient R 2 Between 0.9923 and 0.9997 (table 2), the RSD in day and day is less than 7.7 percent and 17.1 percent, and the recovery rate of the substrate sample in low, medium and high concentrations ranges from 70.0 to 110.2 percent by adding standard (table 3), which shows that the method established by the inventor can be used for analyzing and detecting the central carbon metabolism intermediate in the HEK-293T cell sample.
(3) And (3) determination of the target detection object in the sample to be detected:
with 2-DMBA/d 5 the-2-DMBA is used as a labeling reagent, and is analyzed by combining a liquid chromatography-mass spectrometry combined technology to obtain a target detection object d labeled by the 2-DMBA 5 And (4) substituting the peak area ratio of the internal standard of the 2-DMBA mark into a linear regression equation to calculate the concentration of the target detection object in the sample.
Finally, we tested a small sample of HEK-293T cells (. About.10) using the established assay 3 Individual cells) and finally 21 analytes of interest were detected (table 4).
TABLE 4 chiral carboxylic acids and their content detected in HEK-293T cell samples
As a result, 2-DMBA/d 5 The 2-DMBA can be used as a derivatization reagent to mark central carbon metabolism intermediates with different structures and properties, and by using the strategy, 22 central carbon metabolism intermediates in a HEK-293T cell sample are separated, and accurate quantitative analysis is realized.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (5)
1. A method for quantifying a central carbon metabolism intermediate, the method comprising:
respectively reacting light-weight standard 2-DMBA with the central carbon metabolic intermediate mixed standard substance with different concentrations to obtain a plurality of analytes with different concentrations of standard substances; at the same time, will re-mark d 5 Reacting 2-DMBA with the central carbon metabolic intermediate mixed standard to obtain a derivative of the intermediate as an internal standard;
performing liquid chromatography-mass spectrometry on a plurality of analytes with different concentrations of standard substances and the internal standard substance to obtain different chromatographic peaks and obtaining a linear regression equation through fitting;
reacting light-weight 2-DMBA with a sample to be analyzed, performing liquid chromatography-mass spectrometry after the reaction, and simultaneously adding an internal standard to perform liquid chromatography-mass spectrometry to obtain the peak area and d of a target detection object marked by the 2-DMBA 5 -ratio of peak areas of the 2-DMBA internal standard labeling;
and substituting the ratio into the linear regression equation to calculate the concentration of the target detection object in the sample.
2. The method according to claim 1, wherein in the liquid chromatography-mass spectrometry, the chromatographic separation conditions are as follows: the column used for the liquid phase separation was BEH C18 (2.1X 100mm,1.7 μm, waters.). The column temperature was 50 ℃ and the flow rate was 0.3mL/min. Mobile phases a and B were 2mM ammonium bicarbonate solution and acetonitrile, respectively. The mobile phase gradient is: 0-2 min, 5% B,2-17.5 min, 5% -25% B,17.5-21 min, 25% -90% B,21-24 min, 90% B,24-25 min, 90% -5%,25-30 min, 5%B.
3. The method of claim 1, wherein the reacting the light-labeled 2-DMBA with the different concentrations of the mixed standard of the central carbon metabolic intermediate to obtain a plurality of analytes with different concentrations of the standard comprises:
the central carbon metabolic intermediate mixed standard is formulated into solutions of varying concentrations, including concentrations of 0,0.01,0.025,0.05,0.1,0.25,0.5,1,2.5,5,10,25,50,100,250,500ng/mL, and then labeled with 2-DMBA to obtain a plurality of analytes with standards of varying concentrations.
4. The method of claim 1, wherein the d to be relabeled is 5 Metabolism of 2-DMBA with 1ng/mL central carbonAnd mixing the intermediate with a standard substance for reaction to obtain a derivative of the intermediate as an internal standard.
5. Diazo reagent-DMBA/d 5 Application of-2-DMBA in quantitative analysis of central carbon metabolic intermediates.
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