CN113624862B - Metabolome covering multiple metabolites, metabolic flux analysis method and kit - Google Patents

Metabolome covering multiple metabolites, metabolic flux analysis method and kit Download PDF

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CN113624862B
CN113624862B CN202110790654.9A CN202110790654A CN113624862B CN 113624862 B CN113624862 B CN 113624862B CN 202110790654 A CN202110790654 A CN 202110790654A CN 113624862 B CN113624862 B CN 113624862B
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phosphate
monophosphate
metabolite
diphosphate
acid
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CN113624862A (en
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胡泽平
孟祥骏
庞欢欢
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Tsinghua University
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

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Abstract

The invention discloses a metabolome and metabolic flux analysis method for a plurality of metabolites containing carbonyl, carboxyl and phosphate in a biological sample and a kit used for the method. The method adopts 3-nitrophenylhydrazine to perform derivatization on the metabolite, and then detects the metabolite of carbonyl, carboxyl and phosphate through a liquid chromatography-mass spectrometry combined technology. The method of the invention covers various metabolites containing phosphate groups, and expands the metabolite coverage of the derivatization metabonomics analysis technology. Meanwhile, the invention uses mass spectrum cracking fragments from a derivatization reagent as quantitative fragment ions for metabolic flux analysis, simplifies the metabolic flux analysis process, reduces the workload of metabolic flux analysis and improves the analysis efficiency of metabolic flux research. The kit comprises a reaction device, metabolite standard dry solid powder and a derivatization reagent bag, and can conveniently, quickly and high-flux analyze various metabolites in a biological sample in a metabolome and metabolic flux manner.

Description

Metabolome covering multiple metabolites and metabolic flux analysis method and kit
Technical Field
The invention belongs to the technical field of analysis, and particularly relates to a metabolome covering various metabolites, a metabolic flux analysis method and a kit for the method.
Background
The metabolic research plays an important role in the aspects of the occurrence and development of diseases, the discovery of molecular mechanisms and new therapeutic targets and the like. Metabolomics is an important technology for metabolic studies to comprehensively identify and quantify endogenous small molecule (molecular weight <1,500da) metabolites in biological systems in a high-throughput, unbiased manner. The metabonomics analysis platform based on the liquid chromatography-mass spectrometry (LC-MS) technology has become the most important metabonomics analysis technology due to the advantages of high sensitivity and high specificity. However, due to the wide variety of metabolic species, various physicochemical properties and large concentration difference, and the extremely low abundance of many metabolites with important biological functions or mass spectrum detection signals, the LC-MS technology still has significant technical challenges for comprehensive analysis of metabolites at present, and becomes a bottleneck which seriously restricts metabolic research.
Although chemical derivatization can improve the detection sensitivity of the metabolite, one derivatization reagent can only react with one functional group, so that the coverage of the metabolite by the current derivatization-based metabonomics analysis technology is still very limited.
In view of the disadvantages of insufficient detection sensitivity and less metabolite species coverage in the prior art, it is necessary to develop a new method for metabolome and metabolic flux analysis which can cover a plurality of metabolites, has high detection sensitivity, and is simple and easy to operate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a metabolome and metabolic flux analysis method capable of simultaneously detecting a plurality of metabolites in a biological sample and a detection kit applying the method. The metabolites include metabolites containing carbonyl groups, metabolites containing carboxyl groups and metabolites containing phosphate groups in molecular structures, including but not limited to metabolites such as organic acids, amino acids, sugars, nucleic acids, carnitines and vitamins.
The invention provides a metabolome and metabolic flux analysis method capable of simultaneously detecting a plurality of metabolites in a biological sample, which is realized by the following scheme:
3-nitrophenylhydrazine is adopted as a derivatization reagent to perform derivatization reaction on metabolites in a biological sample to obtain metabolite derivatives, and the LC-MS (liquid chromatography-mass spectrometry) technology is utilized to perform metabolome and metabolic flux analysis. The detection object includes various metabolites including a carbonyl group, a carboxyl group and a phosphate group.
According to an embodiment of the present invention, the metabolite is selected from at least one of a carbonyl group-containing metabolite, a carboxyl group-containing metabolite, and a phosphate group-containing metabolite; preferably a metabolite containing a phosphate group, i.e. a metabolite comprising one or more phosphate groups in the molecular structure.
<xnotran> , 5363 zxft 5363- ,1- ,1- ,1- , 3242 zxft 3242- , 2',3' - -5- , 2- , 2- ,3' - ,3- ,3- ,3- , 5- ,5' - ,5' - ,5' - ,5' - -5- -4- , 5- ,6- ,6- -5' - ,6- ,6- ,6- ,6- ,6- -5' - ,6- ,6- ,6- ,6- -5' - ,6- -5' - ,6- , N- -1- , N- - -6- , N- -9- , O- , </xnotran> <xnotran> O- , O- , O- ,1- (5 '- ) -4- (N- ) -5- , β - , , 2',3'- , -3' - , , , , , , , , , , , -5363 zxft 5363- , , , -5- , -5- , , , -1- , , , -7- , , , , , , , , , , , , , , , , , -5'- , -5' - , , , -2',3' - , -3',5' - , , , -2',3' - , </xnotran> Uridine-3 '-monophosphate, uridine-5' -diphosphate, uridine diphosphate-N-acetylglucosamine, uridine diphosphate galactose, uridine diphosphate glucose, glucose-1,6-diphosphate, glucose-6-phosphate, cyanocobalamine, adenosine triphosphate adenosine, uridine triphosphate, deoxyadenosine triphosphate, adenosine tetraphosphate, aspartyl-4-phosphate, deoxycytidine triphosphate, deoxyuridine Gan Er phosphate, deoxyadenosine monophosphate, adenosine pentaphosphate, adenosine-2 ',3' -cyclic phosphate, adenosine-3 ',5' -cyclic phosphate, nicotinamide ribitol, nicotinamide adenine dinucleotide phosphate, deoxycytidine monophosphate, sphingosine 1 phosphate (D16: 1-P), sphingosine-1-phosphate (D19: 1-P), glycerol-1-stearoyl glycerol phosphate, serine-1-stearoyl glycerol phosphate, xylulose-5-phosphate, erythrose-4-phosphate, glucosamine-6-phosphate, uridine diphosphate-glucuronic acid, gluconic-6-D-lactone, orotic acid, ribose-1-phosphate, inositol-6-phosphate, adenosine-3 ',5' -diphosphate, glycerophosphate.
According to an embodiment of the invention, the biological sample is selected from urine, blood, cerebrospinal fluid, tissue, cells, saliva and stool samples of mammals.
According to an embodiment of the present invention, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide may also be added as a coupling agent to the derivatization reaction.
According to an embodiment of the present invention, a base may be further added to the derivatization reaction as a catalyst, and the base may be an organic base, such as at least one of pyridine, triethylamine, diisopropylethylamine, and N, N-dimethylaminopyridine.
According to an embodiment of the present invention, the assay method can perform metabolic flux analysis on the metabolites concerned using fragment ions specific to the derivatization reagent as the quantification ions, such as carbonyl group-containing metabolites and carboxyl group-containing metabolite derivatives, fragment ions having m/z of 137 as the quantification ions, and fragment ions having m/z of 232 as the quantification ions for phosphate group-containing metabolite derivatives. The method does not need to analyze the mass spectrum cracking behavior of the metabolite, thereby simplifying the metabolic flux analysis process.
According to an embodiment of the invention, the metabolic flux analysis metabolites include a plurality of carbonyl-containing, carboxyl-containing and phosphate-containing metabolites, preferably selected from the group consisting of alpha-ketoglutaric acid, citric acid, fumaric acid, isocitric acid, lactic acid, maleic acid, pyruvic acid, succinic acid, gluconic acid, glucose, 1,3-diphosphoglycerate, galactose 1-phosphate, sphinganine 1-phosphate, glucose 1-phosphate, 2,3-diphosphoglycerate, 2',3' -dideoxyadenosine-5-triphosphate, 2-phosphoglycerate, 2-stearoyl glycerol phosphate, 3 '-adenosine monophosphate, 3-phosphoglycerate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, 5-fluorodeoxyuridine monophosphate, pyridoxamine 5' -phosphate, pyridoxine 5 '-phosphate, pyridoxal 5' -phosphate, ribosyl-5-amino-4-imidazolecarboxamide 5-thymidylate, 6-methylmercaptoguanosine monophosphate, 6-methylmercaptopurine-5 '-ribonucleotide 6-phosphomannose, fructose 6-phosphate, gluconic acid 6-phosphate, sorbitol 6-phosphate, 6-mercaptoxanthine-5' -monophosphate, 6-mercaptoinosine triphosphate, 6-mercaptoguanosine monophosphate, 6-mercaptoinosine-5 '-monophosphate, 6-mercaptopurine riboside-5' -diphosphate, 6-mercaptopurine riboside ribonucleotide 6-mercaptopurine riboside triphosphate, N-acetylglucosamine-1-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetylneuraminic acid-9-phosphate, O-phosphotyrosine, O-phosphoserine, O-phosphothreonine, O-phosphoethanolamine, 1- (5 '-phosphoribosyl) -4- (N-succinylcarboxamide) -5-aminoimidazole, beta-glycerophosphate, carbamyl phosphate, cytidine 2',3 '-cyclic phosphoric acid, cytidine-3' -monophosphate, cytidine diphosphate, guanosine diphosphate, adenosine diphosphate, farnesyl pyrophosphate, glycerophosphocholine, glycerophosphate glycerol, glycerophosphoinositol, glycerophosphoethanolamine, cobalamin, fructose-1,6-diphosphate, reduced nicotinamide adenine dinucleotide phosphate, ribose-5-phosphate, ribulose-5-phosphate, flavin mononucleotide, flavin adenine dinucleotide, inositol-1-phosphate, inosinic acid, mecobalamin, sedoheptulose-7-phosphate, dimethyl phosphate, dihydroxyacetone phosphate, diethyl phosphate, phosphoribosyl pyrophosphate, inositol phosphate, creatine phosphate, methyl phosphate, phosphoserine, phosphoenolpyruvic acid, adenosine phosphate, choline phosphate, phosphatidylserine, phosphatidylethanolamine, thiamine monophosphate, thiamine pyrophosphate, mercaptoxanthine monophosphate, mercaptoguanosine-5 '-diphosphate, mercaptoguanosine-5' -triphosphate, hexa-adenosine phosphate, hexa-inositol phosphate, guanosine-2 ',3' -cyclic phosphate, guanosine-3 ',5' -cyclic phosphate, guanosine triphosphate, guanosine monophosphate, uridine-2 ',3' -cyclic phosphate, uridine-3 '-monophosphate, uridine-5' -diphosphate, uridine diphosphate-N-acetylglucosamine, uridine diphosphate galactose, uridine diphosphate glucose, glucose-1,6-diphosphate, glucose-6-phosphate, cyanocobalamin, adenosine triphosphate, uridine triphosphate, deoxyadenosine triphosphate, adenosine tetraantenoside, aspartyl-4-phosphate, deoxycytidine triphosphate, deoxyuridine Gan Er phosphate, deoxyadenosine monophosphate, penta-adenosine diphosphate, adenosine-2 ',3' -cyclic phosphate, adenosine-3 ',5' -cyclic phosphate, nicotinamide riboside, nicotinamide adenine dinucleotide phosphate, cytidine monophosphate, 1-sphingosine phosphate, sphingosine-1-phosphate (16D: 1-P), sphingosine 1-phosphate (D19: 1-P), 1-stearoyl glycerol phosphate serine, xylulose 5-phosphate, erythrose 4-phosphate, glucosamine-6-phosphate, uridine diphosphate glucuronic acid, 6-phosphogluconate-D-lactone, orotic acid, ribose-1-phosphate, inositol-6-phosphate, L-carnitine, and mixtures thereof, adenosine-3 ',5' -diphosphate, glycerophosphate.
According to an embodiment of the invention, the method of analysis comprises the steps of:
(A) And (3) derivatization reaction: adding a 3-nitrophenylhydrazine solution, a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an alkali solution with the same volume into the metabolite extracting solution, uniformly mixing, and obtaining a metabolite derivative after the reaction is finished;
(B) Metabolome analysis: detecting the metabolites after the derivatization of the 3-nitrophenylhydrazine by using an LC-MS technology;
(C) Metabolic flux analysis: selecting fragment ions specific to a derivatization reagent as quantitative ions of metabolite derivatives during LC-MS data acquisition;
according to an embodiment of the present invention, the solvent used for the solution in step (a) may be an organic solvent, for example, an alcoholic solvent such as at least one of methanol, ethanol, isopropanol;
according to an embodiment of the present invention, the base in step (a) may be an organic base, for example, at least one of pyridine, triethylamine, diisopropylethylamine, N-dimethylaminopyridine; the alkali solution can be an alcohol solution of alkali, and the alcohol can be at least one of methanol, ethanol and isopropanol.
According to an exemplary embodiment of the invention, the analysis method comprises the steps of:
(A1) Collecting a biological sample;
(A2) Centrifuging a biological sample by using a mixed solution of 80% (v/v) methanol and water, and taking supernate to obtain a metabolite extracting solution;
(A3) And (3) derivatization reaction: and (3) sequentially adding 3-nitrophenylhydrazine methanol solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methanol solution and pyridine-methanol solution with the same volume into the metabolite extracting solution in the step (2), uniformly mixing, and obtaining the metabolite derivative after reaction.
(A4) Metabolome analysis: detecting the metabolite after derivatization of the 3-nitrophenylhydrazine by using an LC-MS (liquid chromatography-mass spectrometry) technology;
(A5) Metabolic flux analysis: fragment ions specific to the derivatizing reagent were selected as quantification ions for metabolite derivatives at the time of LC-MS data acquisition.
According to an embodiment of the present invention, no halogenated solvent, preferably no chlorinated solvent, more preferably no dichloromethane, chloroform and/or carbon tetrachloride is used in step (A2).
According to an embodiment of the invention, in step (A3), the concentration of 3-nitrophenylhydrazine is between 5 and 200mM, such as between 1 and 100mM, exemplarily between 35mM;
according to an embodiment of the invention, in step (A3), the concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 5-200mM, such as 10-100mM, exemplarily 21mM;
according to an embodiment of the invention, in step (A3), the pyridine is at a concentration of 0.1-2 (v/v), such as 0.2-1% (v/v), exemplary 0.5% (v/v);
according to an embodiment of the invention, in step (A3), the temperature of the reaction is 0 to 25 ℃, e.g. 2 to 10 ℃, exemplary 4 ℃;
according to an embodiment of the invention, in step (A3), the reaction time is 5-120min, for example 10-60min, exemplary 30min.
According to an embodiment of the present invention, in the step (A5), a fragment ion having m/z (mass-to-charge ratio) of 137 is preferable as the quantitative ion for the carbonyl group-containing metabolite and the carboxyl group-containing metabolite derivative, and a fragment ion having m/z of 232 is preferable as the quantitative ion for the phosphate group-containing metabolite derivative.
According to an embodiment of the present invention, the metabolite is selected from metabolites such as organic acids, amino acids, sugars, nucleic acids, carnitines, and vitamins in the biological sample.
The invention also provides a kit for carrying out the above method. The kit comprises a reaction device, metabolite standard dry solid powder and a derivatization reagent package;
the derivatization reagent bag comprises 3-nitrophenylhydrazine dry solid powder, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide dry solid powder and pyridine-methanol solution;
the reaction device may be a commercially available 48-well plate or 96-well plate suitable for LC-MS detection;
the metabolite standard dry solid powder is obtained by equivalently subpackaging a metabolite standard mixed solution into a plurality of parts and then freeze-drying. One of ordinary skill in the art will appreciate that the standard mix solution used to prepare the metabolite standard dry solid powder contains two different concentrations for the positive control of the derivatization reaction and determination of the metabolite liquid chromatography retention time.
According to an embodiment of the invention, the metabolite standards are selected from one or more of the group consisting of organic acids, amino acids, sugars, nucleic acids, carnitines and vitamin metabolite standards.
According to an embodiment of the invention, the standard may be selected from the group consisting of: <xnotran> 5363 zxft 5363- ,1- ,1- ,1- , 3242 zxft 3242- , 2',3' - -5- , 2- , 2- ,3' - ,3- ,3- ,3- , 5- ,5' - ,5' - ,5' - ,5' - -5- -4- , 5- ,6- ,6- -5' - ,6- ,6- ,6- ,6- ,6- -5' - ,6- ,6- ,6- ,6- -5' - ,6- -5' - ,6- , N- -1- , N- - -6- , N- -9- , O- , O- , O- , </xnotran> <xnotran> O- ,1- (5 '- ) -4- (N- ) -5- , β - , , 2',3'- , -3' - , , , , , , , , , , , -5363 zxft 5363- , , , -5- , -5- , , , -1- , , , -7- , , , , , , , , , , , , , , , , , -5'- , -5' - , , , -2',3' - , -3',5' - , , , -2',3' - , -3'- , -5' - , </xnotran> Uridine-5 ' -diphosphate, uridine diphosphate-N-acetylglucosamine, uridine diphosphate galactose, uridine diphosphate glucose, glucose-1,6-diphosphate, glucose-6-phosphate, cyanocobalamine, adenosine triphosphate, uridine triphosphate, deoxyadenosine triphosphate, adenosine tetraphosphate, aspartyl-4-phosphate, deoxycytidine triphosphate, deoxyurine Gan Er phosphate, deoxyadenosine monophosphate, adenosine pentaphosphate, adenosine-2 ',3' -cyclic phosphate, adenosine-3 ',5' -cyclic phosphate, nicotinamide ribotol, nicotinamide adenine dinucleotide phosphate, deoxycytidine monophosphate, sphingosine 1-phosphate (D16: 1-P), sphingosine-1-phosphate (D19: 1-P), glycerol-1-stearoyl glycerol phosphate, serine-1-stearoyl glycerol phosphate, xylulose-5-phosphate, erythrose-4-phosphate, glucosamine-6-phosphate, uridine diphosphate-glucuronic acid, gluconic-6-acid-D-lactone, orotic acid, ribose-1-phosphate, inositol-6-phosphate, adenosine-3 ',5' -diphosphate, glycerophosphate, alpha-ketoglutaric acid, citric acid, fumaric acid, isocitric acid, lactic acid, maleic acid, pyruvic acid, succinic acid, gluconic acid and/or glucose.
According to the embodiment of the invention, the application method of the derivatization reagent bag is that the 3-nitrophenylhydrazine dry solid powder and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide dry solid powder are accurately weighed and then placed in a glass solvent bottle to be dissolved for use before derivatization reaction. The pyridine content in the pyridine-methanol solution is 0.1-2% (v/v), preferably 0.5% (v/v).
According to an embodiment of the invention, the method of use of the kit comprises the following steps:
(a) Adding the metabolite extracting solution into each hole of the reaction device in equal quantity, and marking as a sample hole;
(b) Dissolving the dry solid powder of the metabolite standard substance by using a solvent, adding the dissolved solid powder into a hole of a reaction device, and marking the dissolved solid powder as a standard substance hole;
(c) Dissolving 3-nitrophenylhydrazine dry solid powder and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide dry solid powder in a solvent, and adding the dissolved dry solid powder and the dissolved dry solid powder into the sample hole and the standard hole in equal amount;
(d) And adding an alkali solution into the sample hole and the standard sample hole, sampling and detecting after the reaction is finished, and adopting the metabolome analysis and the metabolic flux analysis.
According to an embodiment of the present invention, the solvent in step (b) and step (c) may be an organic solvent, for example, an alcoholic solvent, such as at least one of methanol, ethanol, isopropanol;
according to an embodiment of the present invention, the base in step (d) may be an organic base, for example, at least one of pyridine, triethylamine, diisopropylethylamine, N-dimethylaminopyridine; the alkali solution can be an alcohol solution of alkali, and the alcohol can be at least one of methanol, ethanol and isopropanol.
According to an exemplary embodiment of the invention, the method of using the kit comprises the steps of:
(a1) Collecting a biological sample;
(a2) Centrifuging a biological sample by using a mixed solution of 80% (v/v) methanol and water, and taking supernate to obtain a metabolite extracting solution;
(a3) Adding the prepared metabolite extracting solution into each hole of the reaction device in equal quantity, and marking as a sample hole;
(a4) Dissolving the dried solid powder of the metabolite standard substance by using methanol, adding the dissolved metabolite standard substance into a hole of a reaction device, and marking the dissolved metabolite standard substance as a standard substance hole;
(a5) Dissolving 3-nitrophenylhydrazine dry solid powder and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide dry solid powder in methanol, and adding the dissolved solid powder and the dried solid powder into the sample well and the standard well in equal amount;
(a6) And adding pyridine-methanol solution into the sample hole and the standard sample hole, reacting for 5-120min at 0-25 ℃, sampling and detecting, and adopting the metabolome analysis and metabolic flux analysis method.
Advantageous effects
The method of the invention adopts 3-nitrophenylhydrazine to perform derivatization of the metabolite, can simultaneously detect the metabolite containing carbonyl, carboxyl and phosphate groups, and enlarges the coverage range of various components widely existing in the metabolite, thereby having stronger compatibility.
The invention adopts mass spectrum fragmentation fragments derived from a derivatization reagent 3-nitrophenylhydrazine as quantitative fragment ions for metabolic flux analysis. In the existing metabolic flux analysis based on the MRM data acquisition mode, before data acquisition, the mass spectrum cracking behavior of a metabolite needs to be analyzed, the number of isotope labels contained in a mass spectrum cracking fragment of the metabolite is deduced, and thus the mass-to-charge ratio of fragment ions is determined. By applying the method, the mass spectrum cracking behavior of the metabolite does not need to be analyzed, so that the metabolic flux analysis process is simplified, the workload of metabolic flux analysis is reduced, and the analysis efficiency of metabolic flux research is improved.
The kit used in the method is simple and convenient to operate, has high analysis flux, and can greatly improve the working efficiency of metabolic analysis.
Drawings
FIG. 1 shows a general structure of a metabolite containing phosphate groups (R is the other part of the metabolite except the phosphate groups, n.gtoreq.1).
FIG. 2 is a schematic representation of a derivatization kit.
FIG. 3 shows the general reaction scheme of 3-nitrophenylhydrazine derivatized (A) carbonyl, (B) carboxyl and (C) phosphate group metabolites (R is the remaining portion of the metabolite after removal of the carbonyl, carboxyl and phosphate groups).
FIG. 4 is a diagram showing the analysis of the oocyte metabolome principal components.
FIG. 5 is a diagram of the structure of fragment ions involved in metabolic flux analysis.
Figure 6 is a comparison of derivatization versus non-derivatization methods metabolic flux monitoring ion channels.
FIG. 7 is a diagram showing the process of derivatization reaction and a structure of a fragment ion according to the present invention.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 1
In this example it is exemplified that metabolites containing carbonyl, carboxyl and phosphate groups in mouse oocytes are first simultaneously derivatized with 3-nitrophenylhydrazine and then the metabolite derivatives are analyzed using LC-MS techniques.
The general reaction formula of the 3-nitrophenylhydrazine derivatized carbonyl, carboxyl and phosphate groups is shown in FIG. 3:
the specific implementation mode is as follows:
1. instrument for measuring the position of a moving object
Shimadzu LC-30AD high performance liquid chromatograph, sciex QTRAP 6500+ mass spectrometer, equipped with electrospray ion source.
2. Sample preparation
60 oocytes of mice in GV stage and MII stage were collected, placed in 500. Mu.L of 80% (v/v) methanol-water solution, vortexed for 1min, centrifuged at 14000rpm for 15min at 4 ℃, and the supernatant was added to the well of the reaction apparatus.
3. Preparation of metabolite standard solution
Instead of dry solid powder as metabolite standard, 1mL of 80% (v/v) methanol-water solution was added, dissolved by vortexing, and added to the well of the reaction apparatus.
4. Preparation of derivatization reagents
The dry solid powder of 3-nitrophenylhydrazine and the dry solid powder of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide were taken and dissolved in 1mL of methanol each.
5. Sample derivatization
And sequentially adding 50 mu L of 3-nitrophenylhydrazine methanol solution, 50 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methanol solution and 50 mu L of pyridine-methanol solution into the holes added with the metabolite extracting solution and the standard solution, uniformly mixing, and reacting for 30min at the temperature of 4 ℃.
6. Detection method
Mass spectrum conditions: the electrospray ion source adopts a positive and negative ion switching scanning mode, the spraying voltage is 4500V, the ion source temperature is 500 ℃, the atomization gas is 45psi, the gas curtain gas is 25psi, and the desolventizing gas is 45psi.
Chromatographic conditions are as follows: an HSS T3 column (2.1 mm. Times.150mm, 1.8 μm, waters) was used, mobile phase A was 0.03% formic acid-water solution and mobile phase B was 0.03% formic acid-acetonitrile solution. The elution gradient was: 0 to 1.5min,5% by weight of B;35.5-36min,73-90% by weight B;39-39.2min,90-5%B;44min,5% B. The flow rate was 0.3mL/min and the column temperature was 40 ℃.
7. Analysis results
By adopting the analysis method and the kit, the mouse GV-stage and MII-stage oocyte metabolome with the sample amount of only 60 is detected, as shown in figure 4, the principal component analysis shows that the oocyte samples in the same stage are better clustered, and the samples in the two stages can be obviously separated, which shows that the analysis method has extremely high detection sensitivity, and the analysis method and the kit are suitable for metabonomics analysis.
Example 2
In this example, it is illustrated that after derivatization of carbonyl, carboxyl and phosphate group-containing metabolites in HeLa cells with 3-nitrophenylhydrazine, metabolic flux analysis was performed by selecting fragment ions specific to the derivatization reagent as quantification ions at the time of acquisition of metabolic flux data. As shown in FIG. 5, for the carbonyl and carboxyl metabolite derivatives, a fragment ion with m/z of 137 was selected as the quantitative ion, and for the phosphorylated metabolite derivative, a fragment ion with m/z of 232 was selected as the quantitative ion.
The specific implementation mode is as follows:
1. instrument
Shimadzu LC-30AD high performance liquid chromatograph, sciex QTRAP 6500+ mass spectrometer, equipped with electrospray ion source.
2. Sample preparation
Collecting glucose-6-phosphate isomerase wild type and glucose-6-phosphate isomerase knock-out HeLa cells, respectively, placing in 500 μ L80% (v/v) methanol-water solution, vortexing for 1min, centrifuging at 4 deg.C and 14000rpm for 15min, and adding supernatant into the hole of the reaction device.
3. Preparation of metabolite standard solution
Instead of the dry solid powder of the metabolite standard, 1mL of 80% (v/v) methanol-water solution was added, and after vortex dissolution, the solution was added to the well of the reaction apparatus.
4. Preparation of derivatizing reagent
The dried solid powder of 3-nitrophenylhydrazine and the dried solid powder of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide were dissolved in 1mL of methanol each.
5. Sample derivatization
And sequentially adding 50 mu L of 3-nitrophenylhydrazine methanol solution, 50 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methanol solution and 50 mu L of pyridine-methanol solution into the holes added with the metabolite extracting solution and the standard solution, uniformly mixing, and reacting for 30min at the temperature of 4 ℃.
6. Detection method
Mass spectrum conditions: the electrospray ion source adopts a negative ion switching scanning mode, the spraying voltage is 4500V, the ion source temperature is 500 ℃, the atomization gas is 45psi, the air curtain gas is 25psi, and the desolvation gas is 45psi.
Chromatographic conditions are as follows: an HSS T3 column (2.1 mm. Times.150mm, 1.8 μm, waters) was used, mobile phase A was 0.03% formic acid in water and mobile phase B was 0.03% formic acid in acetonitrile. The elution gradient was: 0 to 1.5min,5% by weight of B;35.5-36min,73-90% by weight B;39-39.2min,90-5%B;44min,5% B. The flow rate was 0.3mL/min and the column temperature was 40 ℃.
7. Analysis results
As shown in fig. 6, taking α -ketoglutaric acid as an example, by using a conventional non-derivatization method, before data acquisition, mass spectrometry cracking behavior of α -ketoglutaric acid needs to be studied first, so as to infer the number of isotopic labels contained in fragment ions of each isotopologue after mass spectrometry cracking, and then LC-MS data acquisition is performed by using the fragment ions as quantitative ions; after the derivatization method is adopted, each isotopologue can generate the same fragment ions derived from 3-nitrophenylhydrazine, the fragment ions are used as quantitative ions, the number of isotopes contained in the fragment ions does not need to be inferred, and the number of ion channels needing to be monitored can be reduced. Table 1 shows the results of isotopic distribution of metabolites in two groups of HeLa cells, each group containing 5 biological replicates.
TABLE 1 results of isotopic distribution of metabolites in two groups of HeLa cells
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Figure BDA0003160739320000151
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Figure BDA0003160739320000161
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims (13)

1. A can detect the metabolome and metabolic flux analytic method of multiple metabolite in the biological sample at the same time, characterized by that, use 3-nitrophenylhydrazine as derivatization reagent to carry on the metabolome and metabolic flux analysis with the liquid chromatography-mass spectrometry technology after the metabolite in the biological sample is derivatized;
wherein, the specific fragment ions of the derivatization reagent are used as quantitative ions to detect and analyze the metabolic flux of the related metabolites;
the biological sample is selected from the group consisting of urine, blood, cerebrospinal fluid, tissue, cells, saliva, and stool samples of a mammal;
1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is also added in the derivatization reaction as a coupling agent;
adding alkali as a catalyst in the derivatization reaction, wherein the alkali is at least one of pyridine, triethylamine, diisopropylethylamine and N, N-dimethylaminopyridine;
the metabolites are carbonyl-containing metabolites, carboxyl-containing metabolites and phosphate-containing metabolites; the quantitative ions are specific fragment ions of a derivatization reagent 3-nitrophenylhydrazine;
the fragment ion with m/z of 137 was used as the quantitative ion for the carbonyl group-containing metabolite derivative and the carboxyl group-containing metabolite derivative, and the fragment ion with m/z of 232 was used as the quantitative ion for the phosphate group-containing metabolite derivative.
2. The analytical method according to claim 1, wherein the metabolite is at least one selected from the group consisting of an organic acid, an amino acid, a sugar, a nucleic acid, a carnitine, and a vitamin metabolite.
3. The assay of claim 1, comprising the steps of:
(A) And (3) derivatization reaction: adding 3-nitrophenylhydrazine solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and alkali solution with the same volume into the metabolite extracting solution, uniformly mixing, and obtaining a metabolite derivative after reaction is finished;
(B) Metabolome analysis: detecting the metabolites after the derivatization of the 3-nitrophenylhydrazine by using a liquid chromatography-mass spectrometry combined technology;
(C) Metabolic flux analysis: selecting specific fragment ions of a derivatization reagent as quantitative ions of metabolite derivatives during data acquisition of liquid chromatography-mass spectrometry;
the solvent used in the solution in the step (A) is an alcohol solvent;
the base in step (a) is an organic base; the alkaline solution is an alcohol solution of an alkali, and the alcohol is at least one of methanol, ethanol and isopropanol.
4. The analytical method according to claim 3, wherein the alcohol solvent in step (A) is at least one selected from the group consisting of methanol, ethanol and isopropanol.
5. The analytical method according to claim 3, wherein the organic base in step (A) is at least one selected from the group consisting of pyridine, triethylamine, diisopropylethylamine, and N, N-dimethylaminopyridine.
6. An analytical method as claimed in claim 1 or 2, comprising the steps of:
(A1) Collecting a biological sample;
(A2) Centrifuging the biological sample with 80% (v/v) methanol-water solution, and collecting supernatant to obtain metabolite extract;
(A3) And (3) derivatization reaction: adding 3-nitrophenylhydrazine methanol solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methanol solution and pyridine-methanol solution with the same volume into the metabolite extracting solution in the step (A2) in sequence, mixing uniformly, and obtaining a metabolite derivative after the reaction is finished;
(A4) Metabolome analysis: detecting the metabolite after derivatization of the 3-nitrophenylhydrazine by using a liquid chromatography-mass spectrometry combined technology;
(A5) Metabolic flux analysis: when the liquid chromatography-mass spectrometry data are collected, specific fragment ions of a derivatization reagent are selected as quantitative ions of metabolite derivatives for metabolic flux analysis.
7. The assay method according to claim 6, wherein in step (A3), the concentration of the 3-nitrophenylhydrazine is from 5 to 200mM;
the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 5-200mM.
8. The assay of claim 6, wherein in step (A3), the pyridine is present at a concentration of 0.1-2% (v/v);
the reaction temperature is 0-25 ℃;
the reaction time is 5-120min.
9. The assay method according to any one of claims 1 to 5, wherein the phosphate group-containing metabolite is a metabolite comprising one or more phosphate groups in the molecular structure.
10. The assay of claim 1, wherein the phosphate group-containing metabolite is selected from the group consisting of 1,3-diphosphoglycerate, galactose-1-phosphate, sphinganine-1-phosphate, glucose-1-phosphate, 2,3-diphosphoglycerate, 2',3' -dideoxyadenosine-5-triphosphate, 2-phosphoglycerate, 2-stearoyl glycerol phosphate, 3' -adenosine monophosphate, 3-glycerol phosphate, glyceraldehyde 3-phosphate, 5-fluorodeoxyuridine monophosphate, pyridoxamine 5' -phosphate, pyridoxine 5' -phosphate, pyridoxal 5' -phosphate, ribosyl-5-amino-4-imidazolecarboxamide 5-thymidylate, 6-methylthioguanosine monophosphate, 6-methylthiopurine-5 ' -monophosphate ribonucleotide 6-phosphomannose, 6-phosphofructose, 6-phosphogluconic acid, 6-sorbitol phosphate, 6-mercaptoxanthine-5 ' -monophosphate, 6-mercaptoinosine triphosphate, 6-mercaptoguanosine monophosphate, 6-mercaptoguanylic acid, 6-mercaptoinosine-5 ' -monophosphate, 6-mercaptopurine ribonucleoside-5 ' -diphosphate, 6-mercaptopurine ribonucleoside triphosphate, N-acetylglucosamine-1-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetylneuraminic acid-9-phosphate, N-acetylneuraminic acid-5 ' -monophosphate, N-thioglycoside triphosphate, N-acetylglucosyl-1-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetylneuraminic acid-9-phosphate, and mixtures thereof, <xnotran> O- , O- , O- , O- ,1- (5 ' - ) -4- (N- ) -5- , β - , , 2',3' - , -3' - , , , , , , , , , , , -5363 zxft 5363- , , , -5- , -5- , , , -1- , , , -7- , , , , , , , , , , , , , , , , , -5' - , -5' - , , , -2',3' - , -3',5' - , , , -2', </xnotran> 3 '-cyclic phosphate, uridine-3' -monophosphate, uridine-5 '-diphosphate, uridine diphosphate-N-acetylglucosamine, uridine diphosphate galactose, uridine diphosphate glucose, glucose-1,6-diphosphate, glucose-6-phosphate, cyanocobalamin, adenosine triphosphate, uridine triphosphate, deoxyadenosine triphosphate, adenosine tetraphosphate, aspartyl-4-phosphate, deoxycytidine triphosphate, deoxyurine Gan Er phosphate, deoxyadenosine monophosphate, adenosine pentaphosphate, adenosine-2', 3 '-cyclic phosphate, adenosine-3', 5 '-cyclic phosphate, nicotinamide ribitol, nicotinamide adenine dinucleotide phosphate, deoxycytidine monophosphate, sphingosine 1-phosphate (D16: 1-P), sphingosine 1-phosphate (D19: 1-P), glycerol 1-stearoyl glycerol phosphate, serine 1-stearoyl glycerol phosphate, xylosine 5-monophosphate, D6-fructose 4-diphosphate, erythrosine 6-fructose 6-diphosphate, inositol 1' -monophosphate, inositol 6-diphosphate, inositol 6-phosphate, glucose-6-phosphate, inositol 1 '-monophosphate, inositol diphosphate, inositol 6-D6-phosphate, and D19' -monophosphate.
11. The assay of claim 1, wherein the metabolite is selected from the group consisting of alpha-ketoglutaric acid, citric acid, fumaric acid, isocitric acid, lactic acid, maleic acid, pyruvic acid, succinic acid, gluconic acid, glucose, 1,3-diphosphoglycerate, galactose 1-phosphate, sphinganine 1-phosphate, glucose 1-phosphate, 2,3-diphosphoglycerate, 2',3' -dideoxyadenosine-5-triphosphate, 2-phosphoglycerate, 2-stearoyl glycerol phosphate, 3' -adenosine monophosphate, 3-phosphoglycerate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, 5-fluorodeoxyuridine monophosphate, pyridoxamine 5' -phosphate, pyridoxine 5' -phosphate, pyridoxal 5' -phosphate, ribosyl-5-amino-4-imidazolecarboxamide 5-thymidylate, 6-methylmercaptoguanosine monophosphate, 6-methylmercaptopurine-5 ' -ribonucleotide 6-phosphomannose, fructose 6-phosphate, gluconic 6-phosphate, sorbitol 6-phosphate, 6-mercaptoxanthine-5 ' -monophosphate, 6-mercaptoinosine triphosphate, 6-mercaptoguanosine monophosphate, 6-mercaptoinosine-5 ' -monophosphate, 6-mercaptopurine riboside-5 ' -diphosphate, 6-mercaptopurine ribonucleoside triphosphate, N-acetylglucosamine 1-acetylglucose phosphate, pyridoxamine-5 ' -monophosphate, N-acetyl-glucosamine-6-phosphate, N-acetylneuraminic acid-9-phosphate, O-phosphotyrosine, O-phosphoserine, O-phosphothreonine, O-phosphoethanolamine, 1- (5 ' -phosphoribosyl) -4- (N-succinylcarboxamide) -5-aminoimidazole, beta-glycerophosphate, carbamoyl phosphate, cytidine 2',3' -cyclic phosphoric acid, cytidine-3 ' -monophosphate, cytidine diphosphate, guanosine diphosphate, adenosine diphosphate, farnesyl pyrophosphate, glycerophosphocholine, glycerophosphoinositol, glycerophosphoethanolamine, cobalamin, fructose-1,6-diphosphate, reduced nicotinamide adenine dinucleotide phosphate, ribose-5-phosphate, ribulose-5-phosphate, flavin mononucleotide, flavin adenine dinucleotide, inositol-1-phosphate, inosinic acid, mecobalamin, sedoheptulose-7-phosphate, dimethyl phosphate, dihydroxyacetone phosphate, diethyl phosphate, ribose pyrophosphate, inositol phosphate, creatine phosphate, methyl phosphate, serine phosphate, phosphoenolpyruvic acid, adenosine phosphate, choline phosphate, phosphatidylserine, phosphatidylethanolamine, thiamine monophosphate, thioguanine pyrophosphate, thioguanine monophosphate, thioguanine guanosine 5' -diphosphate, guanosine 5' -triphosphate, hexaadenosine phosphate, hexakisphosphate, hexakis-2 ', <xnotran> 3'- , -3',5'- , , , -2',3'- , -3' - , -5'- , -5' - , -N- , , , -5363 zxft 5363- , -6- , , , , , , , -4- , , 3242 zxft 3242 , , , -2',3' - , -3',5' - , , , , ,1- ,1- (d 16: 1-P), 1- (d 19: 1-P), 1- ,1- , -5- , -4- , -6- , - ,6- -D- , ,1- ,6- , -3',5' - , </xnotran> At least one of glycerophosphoric acid.
12. Use of a kit for covering a plurality of metabolites of a metabolome and metabolic flux analysis method, the kit comprising a reaction device, a metabolite standard dry solid powder, and a derivatizing agent package;
the derivatization reagent bag comprises 3-nitrophenylhydrazine dry solid powder, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide dry solid powder and pyridine-methanol solution;
the metabolite standard dry solid powder is powder obtained by equivalently subpackaging a metabolite standard mixed solution into a plurality of parts and then freeze-drying;
the metabolite standard is selected from at least one of the following components: 5363 Zxft 5363-diphosphoglycerate, galactose-1-phosphate, sphinganine-1-phosphate, glucose-1-phosphate, 2,3-diphosphoglycerate, 2',3' -dideoxyadenosine-5-triphosphate, 2-phosphoglycerate, 2-stearoyl glycerol phosphate, 3' -adenosine monophosphate, 3-glycerol phosphate, 3-phosphoglycerate, 5-fluorodeoxyuridine monophosphate, pyridoxamine 5' -phosphate, pyridoxine 5' -phosphate, pyridoxal 5' -phosphate, ribosyl-5-amino-4-imidazolecarboxamide 5-thymidylate, 6-methylthioguanosine monophosphate, 6-methylthiopurine-5 ' -monophosphate ribonucleotide, 6-sorbitol phosphate, 6-mercaptoxanthine-5 ' -monophosphate 6-mercaptoinosine triphosphate, 6-mercaptoguanosine monophosphate, 6-mercaptoguanylic acid, 6-mercaptoinosine-5 ' -monophosphate, 6-mercaptopurine ribonucleoside-5 ' -diphosphate, 6-mercaptopurine ribonucleoside triphosphate, N-acetylglucosamine-1-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetylneuraminic acid-9-phosphate, O-phosphotyrosine, O-phosphoserine, O-phosphothreonine, O-phosphoethanolamine, 1- (5 ' -phosphoribosyl) -4- (N-succinylcarboxamide) -5-aminoimidazole, and pharmaceutically acceptable salts thereof, beta-glycerophosphate, carbamoyl phosphate, cytidine 2',3' -cyclic phosphate, cytidine 3' -monophosphate, cytidine diphosphate, guanosine diphosphate, farnesyl pyrophosphate, glycerophosphate glycerol, glycerophosphoinositide, glycerophosphoethanolamine, cobalamin, reduced nicotinamide adenine dinucleotide phosphate, flavin mononucleotide, flavin adenine dinucleotide, inositol-1-phosphate, inosinic acid, mecobalamin, sedoheptulose-7-phosphate, dimethyl phosphate, diethyl phosphate, inositol phosphate, methyl phosphate, phosphoserine, phosphoenolpyruvic acid, choline phosphate, phosphatidylserine, phosphatidylethanolamine, thiamine monophosphate, thiamine pyrophosphate, mercaptoxanthine monophosphate, thioguanine-5 ' -diphosphate, thioguanine-5 ' -triphosphate, hexaadenosine monophosphate, inositol hexaphosphate, guanosine 2',3' -cyclic phosphoric acid, guanosine-3 ',5' -cyclic phosphoric acid, uridine-2 ',3' -cyclic phosphoric acid, uridine-3 ' -monophosphate, uridine-5 ' -diphosphate, uridine-diphosphate-N-acetylglucosamine, uridine diphosphate galactose, uridine diphosphate glucose, glucose-1,6-diphosphate, cyanocobalamine, adenosine triphosphate, uridine triphosphate, deoxyadenosine triphosphate, tetradenosine-4-phosphate, deoxycytidine triphosphate, deoxyurine Gan Er phosphate, deoxyadenosine monophosphate, adenosine pentaphosphate, adenosine diphosphate, uridine-2 ',3' -cyclic phosphate, uridine-3 ' -monophosphate, uridine-5 ' -diphosphate, uridine-diphosphate-N-acetylglucosamine, uridine diphosphate, uridine-4-phosphate, deoxyadenosine triphosphate, deoxyurea Gan Er phosphate, deoxyadenosine monophosphate, adenosine diphosphate, deoxyadenosine monophosphate, uridine-2 ',3' -monophosphate, uridine-triphosphate, deoxyadenosine monophosphate, and the like, adenosine-2 ',3' -cyclic phosphate, adenosine-3 ',5' -cyclic phosphate, nicotinamide adenine dinucleotide phosphate, deoxycytidine monophosphate, sphingosine-1-phosphate (D16: 1-P), sphingosine-1-phosphate (D19: 1-P), glycerol-1-stearoyl glycerophosphate, serine-1-stearoyl glycerophosphate, xylulose-5-phosphate, erythrose-4-phosphate, glucosamine-6-phosphate, uridine diphosphate-glucuronic acid, 6-phosphogluconate-D-lactone, ribose-1-phosphate, inositol-6-phosphate, adenosine-3 ',5' -diphosphate, glycerophosphate,
wherein the analysis method comprises the following steps:
(A1) Collecting a biological sample;
(A2) Centrifuging a biological sample by using a mixed solution of 80% (v/v) methanol and water, and taking supernate to obtain a metabolite extracting solution;
(A3) And (3) derivatization reaction: sequentially adding 3-nitrophenylhydrazine methanol solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methanol solution and pyridine-methanol solution with the same volume into the metabolite extracting solution in the step (2), uniformly mixing, and obtaining a metabolite derivative after reaction;
(A4) Metabolome analysis: detecting the metabolites after the derivatization of the 3-nitrophenylhydrazine by using an LC-MS technology;
(A5) Metabolic flux analysis: selecting fragment ions specific to a derivatization reagent as quantitative ions of metabolite derivatives during LC-MS data acquisition,
wherein, for the metabolite containing carbonyl and the metabolite derivative containing carboxyl, the fragment ion with m/z of 137 is selected as the quantitative ion, and for the metabolite derivative containing phosphate, the fragment ion with m/z of 232 is selected as the quantitative ion.
13. Use according to claim 12, wherein the pyridine-methanol solution has a pyridine content of 0.1-2% (v/v).
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