CN116430026A - Nucleotide analysis method based on pairing derivatization technology - Google Patents
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- CN116430026A CN116430026A CN202310477971.4A CN202310477971A CN116430026A CN 116430026 A CN116430026 A CN 116430026A CN 202310477971 A CN202310477971 A CN 202310477971A CN 116430026 A CN116430026 A CN 116430026A
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Abstract
The invention relates to the technical field of analysis and detection. More particularly, the present invention relates to a method of analyzing nucleotides based on a pairing derivatization technique. 4-ethylbenzylamine 4-EBA and 4-methylbenzylamine 4-MBA are used as derivatization reagents in detecting nucleotide; the method for determining the nucleotide quantification improves accuracy and precision, and the analysis strategy can achieve the effect similar to that of using the isotope internal standard although the isotope internal standard is not used.
Description
Technical Field
The invention relates to the technical field of analysis and detection. More particularly, the present invention relates to a method of analyzing nucleotides based on a pairing derivatization technique.
Background
Nucleotides are an important biological molecule consisting of nitrogen groups, five carbon sugars and phosphate groups. They are distributed in the nucleus and cytoplasm of organs, tissues and cells of the body asThe nucleic acid component participates in basic life activities such as inheritance, growth, development and the like of organisms. In addition, there are a considerable number of free nucleotides in organisms that are involved in many critical cellular processes, energy metabolism and signal transduction, among others. The analysis of the nucleotide level can provide information for solving the relationship between the nucleotide and the disease, and has great significance for the elucidation and clinical diagnosis and treatment of the pathogenesis of the related disease. Currently, a number of detection methods have been developed and used for nucleotide analysis. Such as Nuclear Magnetic Resonance (NMR) [1] Capillary Electrophoresis (CE) [2] High Performance Liquid Chromatography (HPLC) [3] Capillary electrophoresis mass spectrum (CE-MS) [4] And liquid chromatography mass spectrometry (LC-MS) [5] . The LC-MS has the characteristics of high specificity, good separability, high sensitivity and the like, and is the most powerful platform for analyzing nucleotide at present.
However, LC-MS detection of nucleotides is also required to solve the following problems: (1) The phosphate group of the nucleotide can be combined with liquid-phase steel, a mass spectrum spray needle and the like, so that chromatographic peak tailing is caused, and the sensitivity is reduced. The action may be complexation, oxidation or epoxidation [6] The method comprises the steps of carrying out a first treatment on the surface of the (2) The nucleotide polarity is large, so that the retention force on the reversed phase chromatographic column is weak, and the chromatographic separation of the nucleotide is not facilitated. The ionization efficiency of the ion source in the electrospray ion source is poor, so that the mass spectrum detection sensitivity of the ion source is poor; (3) In addition, LC-MS is typically quantitatively analyzed using stable isotope internal standards in order to accurately quantify target analytes, minimizing the matrix effects of target analytes in detection. However, isotopic internal standards are difficult to obtain or very expensive, and development of corresponding methods is urgently needed to solve this problem.
To solve the above problems, derivatization techniques are generally employed; derivatization techniques quantitatively convert a target compound in a sample that is difficult to assay to another compound that is easy to assay by chemical reaction, and by the latter assay can be used to perform qualitative or quantitative analysis of the target compound. Currently, derivatizing reagents for mass spectrometry of nucleotides are: 3-Aminomethylpyridine (AMPy), trimethylsilylated diazomethane (TMSD), N- (tert-butyldimethylsilyl) -N-Methyltrifluoroacetamide (MTBSTIA), 2- (diazomethyl) -N-methyl-N-phenylBenzamide (2-DMBA) and the like. In an analytical method in which the nucleotide is determined based on chemically derivatized LC-MS/MS, 3-aminomethylpyridine (AMPy) [7] Is used to derivatize the nucleotides, but the product is not retained on the reverse phase chromatography column. Li et al. [8] The nucleotide in a549 and HCT116 cells was determined using a trimethylsilylated diazomethane (TMSD) derivatization strategy. In addition to this, there are studies [9] N- (tert-butyldimethylsilyl) -N-Methyltrifluoroacetamide (MTBSTFA) derivatization was developed to determine the nucleotide in cells, and after reaction of these derivatizing reagents with the nucleotide, the product was retained on a reverse phase chromatographic column, but none of these methods provided a one-to-one internal standard. Liu et al. [10] A pair of light and heavy stable isotope labeling reagents, 2- (diazomethyl) -N-methyl-N-phenyl-benzamide (2-DMBA) and d, were synthesized 5 -2- (diazomethyl) -N-methyl-N-phenyl-benzamide (d) 5 -2-DMBA) to provide a one-to-one internal standard, they used this method to perform sensitive, accurate ribonucleic acid detection on 8 cells. On the basis of this, they [11] Another pair of light and heavy stable isotope labeling reagents, 2- (diazomethyl) phenyl) (9-methyl-1, 3,4, 9-tetrahydro-2 h-pyridine [3,4-b ], is synthesized]Indol-2-yl) methane (DMPI) and d 3 - (2- (diazomethyl) phenyl) (9-methyl-1, 3,4, 9-tetrahydro-2 h-pyridine [3, 4-b)]Indol-2-yl) methane (d) 3 -DMPI). In this way, they determined the nucleotides of individual mammalian cells. However, the synthesis of these reagents is complicated and cumbersome, limiting their use.
In the present invention, we optimized 4-methylbenzylamine (4-MBA) as a highly efficient pre-column derivatization reagent, with 4-ethylbenzylamine (4-EBA) as the derivatization nucleotide as a one-to-one internal standard. A4-MBA/4-EBA based pairing derivatization method was developed that was accurate, sensitive, and simultaneously quantified for 12 nucleotides by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
Disclosure of Invention
Object of the Invention
The invention provides a pairing derivatization strategy based on 4-MBA/4-EBA, which has the advantages of avoiding isotope use and realizing high-sensitivity and high-selectivity nucleotide LC-MS/MS analysis method.
Technical proposal
The present invention solves the above-mentioned problems by adopting a technical scheme as shown in fig. 1:
4-ethylbenzylamine 4-EBA and 4-methylbenzylamine 4-MBA are used as derivatization reagents for detecting free nucleotide content in cells or tissues.
The biological sample is subjected to protein precipitation by taking a product obtained after the reaction of 4-ethylbenzylamine (4-EBA) and 12 nucleotide standards as an internal standard, free nucleotides in the supernatant are chemically marked by 4-methylbenzylamine (4-MBA), and after mixing, detection and analysis are performed by using LC-MS/MS. It comprises the following steps:
(1) The volume ratio is 1:1 acetonitrile and water are used as solvents to prepare a nucleotide standard substance solution;
(2) The volume ratio is 1:1 acetonitrile and water are used as solvents, EDC, 4-EBA and NMM are sequentially added and uniformly mixed to obtain 4-EBA derivative solution;
(3) Preparing a nucleotide mixed standard solution from the nucleotide mother solution obtained in the step (1), adding a 4-EBA derivatization solution, uniformly mixing, and reacting to obtain a derivatization internal standard solution;
(4) The volume ratio is 1: acetonitrile and water as solvent, EDC, 4-MBA and NMM are added in sequence and evenly mixed to obtain 4-MBA derivatization solution;
(5) Drawing a standard curve: selecting a nucleotide standard substance, diluting the nucleotide standard substance into a series of standard solutions with a certain concentration by using a solvent, adding the 4-MBA derivatization solution for derivatization reaction, adding the internal standard solution obtained in the step (3), and diluting to obtain solutions to be tested of all standard curves;
(6) Detecting the solution to be detected of each standard curve by adopting an LC-MS/MS method, and establishing a standard curve of each nucleotide; wherein, the liquid chromatography conditions are as follows: chromatographic column: shimadzu Shim-pack GIST C18 column (2.1X100 mm,2.0 μm); flow rate: 0.3mL/min; column temperature: 35 ℃; sample injection amount: 4. Mu.L; mobile phase: a is 5mM NH 4 HCO 3 And adjusting the pH to 7.8, B being pure acetonitrile; elution gradient: 0-3min,5% B;3-7min,5% -20% B;7-10min,20% -25% B;10-15min,25-30% b;15-20min,30% -60% B;20-22min,60% -80% B;22-24min,80% B;24-26min,80% -5% B; then equilibrated for 4min.
The mass spectrum conditions are as follows: adopting a positive ion mode, wherein the atomized air flow is 3L/min; the flow rate of the drying gas and the heating gas is 10L/min, the interface temperature is 250 ℃, the temperature of the DL tube is 250 ℃, and the temperature of the heating block is 400 ℃. Multiplex Reaction Monitoring (MRM) mode is used to detect post-derivatization nucleotides.
(7) Extracting a biological sample and precipitating proteins, and drying the obtained supernatant with nitrogen to obtain a sample A;
(8) Dissolving the sample A obtained in the step (7) in a volume ratio of 1:1, adding 4-MBA derivatization solution into acetonitrile and an aqueous solvent, uniformly mixing and reacting until the nucleotide reaction is completed, adding the derivatization internal standard solution obtained in the step (3), diluting with water, uniformly mixing, and centrifuging to obtain a supernatant sample B.
(9) And (3) carrying out reversed-phase liquid chromatography-electrospray mass spectrometry on the supernatant sample B obtained in the step (8), and carrying out quantitative analysis on the nucleotide.
Wherein in step (4), the derivatization temperature, derivatization time, and the molar ratio of 4-MBA, EDC, and NMM to nucleotides are optimized to obtain good labeling efficiency (as shown in fig. 2). First, the reaction temperature (20 to 90 ℃) is optimized. In our results, the peak area of the derivative reached a maximum at 60 ℃. The reaction time was then optimized (0.5 h-7 h). From 0.5 to 4h, the peak area of the derivative increases with time, and becomes stable after 4 h. Thus 4h was chosen as the optimal reaction time. The molar ratio of substrate to 4-MBA was then optimized from 1:1000 to 1:50000. Peak area reached a maximum at 1:5000. Thus, a molar ratio of 1:5000 was chosen. The optimum concentration of EDC was then examined. The molar ratio optimization range of the substrate to EDC is set to be 1:1000-1:50000, and the molar ratio of 1:5000 is also selected. Finally, the optimized range of the molar ratio of the substrate to NMM is set to be 1:1000-1:50000, and the peak area of the derivative is less in change under different molar ratios. Thus, the molar ratio of substrate to NMM was chosen to be 1:1000. In general, the molar ratios of 4-MBA, EDC and NMM to nucleotides were set to 5000, 5000 and 1000, respectively, by 4-MBA reaction at 60℃for 4 h. Under these conditions, the derivatization reaction was more complete (FIG. 3). In step (2), the reaction of 4-EBA is carried out under such optimized reaction conditions.
The biological sample is a tissue or a cell.
Deproteinizing the tissue in step (7): adding the tissue into a 2mL centrifuge tube, adding precooled methanol and mixed solution with water volume ratio of 4:1, grinding in a grinder for 60sec each time, grinding for 10 times at intervals of 10sec each time, centrifuging the homogenate at 13000rpm and 4 ℃ for 10min, and taking out supernatant.
Deproteinization of cells in step (7): washing cells with PBS buffer solution, placing into a 1.5mL centrifuge tube, centrifuging at 3000rpm for 5min to precipitate cells, discarding supernatant, adding precooled mixed solution of methanol and water with water volume ratio of 4:1, incubating at 4deg.C for 30min, vortex mixing, centrifuging at 13000rpm at 4deg.C for 10min, and taking out supernatant.
In the step (8), in the nucleotide LC-MS/MS analysis method based on the pairing derivatization technology, the volume ratio of the biological sample A to the internal standard reaction solution to the water is 1:1:1.
principle of this reaction (formula below):
although there are many reports of quantitative nucleotide analysis using derivatization strategies in combination with liquid analysis, the limitation of the isotope internal standard is still a challenge for analyzing nucleotide, and compounds similar to the structure of the target compounds can be used as substitutes of the isotope internal standard.
In the present invention, we propose for the first time to quantitatively analyze nucleotides using readily available 4-MBA or 4-EBA as a paired derivatizing reagent. Homologs 4-MBA and 4-EBA with similar structures are used as paired derivatization reagents, and react with nucleotides under the action of condensing agent EDC and alkali and catalyst NMM to remove one molecule H from 4-MBA or 4-EBA and nucleotides 2 O, forming a phosphoramide structure. Because the derivatization reagent contains benzene ring, the hydrophobicity of the nucleotide can be increased, and the nucleotide is revealedSignificantly improving their retention behavior in reverse phase chromatography columns to reduce interferences. On the other hand, due to the existence of amino in the 4-MBA or 4-EBA structure, the ionization efficiency of the 4-MBA or 4-EBA modified nucleotide in an ESI source is increased, so that the sensitivity of mass spectrum detection of the nucleotide is remarkably improved. 4-EBA-derived nucleotide mixed standard solution is used as a derived internal standard solution; the free nucleotide of the 4-MBA derivatization sample (biological samples such as cells or tissues are treated to obtain supernatant, 4-MBA is added for derivatization), the content of the free nucleotide in the supernatant of the sample is analyzed by LC-MS/MS (the peak area of the 4-MBA modified nucleotide/the peak area of the 4-EBA modified nucleotide is calculated to obtain a relative result), the calculation can reduce the matrix effect of the quantitative analysis of the nucleotide in the sample.
The calculation formula is as follows: relative value = 4-MBA modified nucleotide peak area/4-EBA modified nucleotide peak area.
4-MBA and 4-EBA derivatized nucleotide reaction formula
Advantageous effects
(1) No report is made on the use of 4-MBA or 4-EBA as a derivatizing agent, and the inventor discovers the reagent for the first time. The derivatization reaction marking efficiency of the invention is 81.5% -100%, and the efficiency of the derivatization reaction marking is Sheng et al. [7] The labeling efficiency using 3-aminomethylpyridine (AMPy) was 74.4% to 89.3%. The derivatization reaction related by the invention has higher marking efficiency, and is favorable for quantitative analysis.
(2) The derivatization reaction can obviously improve the retention of the nucleotide on a reversed-phase chromatographic column and improve the mass spectrum response, (figure 4), namely, the quantitative limit of the nucleotide before the reaction is 0.0333 to 1.6640pmol, the quantitative limit of the nucleotide marked by 4-MBA is 0.0002 to 0.0166pmol, and the sensitivity after the reaction is improved by 10.0 to 166.4 times. As biological samples are difficult to obtain, the method is applied to 30mg colon tissue and 800 mu L of extraction solvent is addedExtracting and taking 400. Mu.L of supernatant to quantify nucleotide has been reported in the literature [12] When not derivatizing, 50mg of tissue should be added with 500. Mu.L and 400. Mu.L of supernatant should be taken to quantify the nucleotides, so the method requires a small sample size.
(3) The pairing derivatization strategy related by the invention has the advantages that the derivatization reagents 4-MBA and 4-EBA are a pair of homologs, only differ by 1 '-CH 2' group, the nucleotide standard substance reacts with the 4-EBA and is added into a sample to be detected for derivatization of the 4-MBA as an internal standard, so that each homolog to be detected has one-to-one correspondence as an internal standard, the purpose of reducing matrix effect is achieved (figure 5), and the accuracy and precision of nucleotide quantification can be effectively improved, and Li et al. [8] Derivatization strategy with trimethylsilylated diazomethane (TMSD) using ATP- 13 C 10 , 15 N 5 And AMP- 13 C 10 , 15 N 5 The internal standard of nucleotide quantification is that the daily internal precision and the daytime precision are respectively 2.1 to 14.7 percent and 3.9 to 14.7 percent; the accuracy in the day and the accuracy in the daytime are-12.0% -13.5%; the matrix effect is 91.9% -110.9%. Zhang et al. [9] Derivatization of nucleotide strategies with N- (tert-butyldimethylsilyl) -N-Methyltrifluoroacetamide (MTBSTFA), likewise using ATP- 13 C 10 , 15 N 5 And AMP- 13 C 10 , 15 N 5 The internal standard of nucleotide quantification is that the daily precision and the daytime precision are respectively 1.3 to 14.4 percent and 3.4 to 14.8 percent, the daily accuracy and the daytime accuracy are respectively-10.8 to 9.4 percent and-10.4 to 11.2 percent, and the matrix effect is 91.1 to 113.9 percent. In the invention, the paired derivatization strategy is utilized to analyze the nucleotide in the colon and the cell, in the quantitative methodology of investigating the colon tissue, the daily precision and the daytime precision are respectively 2.3-13.7% and 0.9-13.8%, the daily accuracy and the daytime accuracy are respectively-7.3-10.3% and-9.4-6.9%, and the matrix effect is 87.2-114.6%; in the method for examining cell quantification, the daily precision and the daytime precision are respectively 1.4-14.0% and 2.6-11.9%, the daily accuracy and the daytime accuracy are respectively-11.0-10.7% and-11.2-12.2%, and the matrix effect is achieved86.3 to 115.4 percent. It can be seen that the use of the assay strategy of the present invention, although without the use of an isotopic internal standard, achieves a similar effect as the use of an isotopic internal standard.
Drawings
FIG. 1 is a technical roadmap of the invention;
FIG. 2 is a graph showing peak areas of products obtained by derivatizing nucleotides under different conditions in the present invention (A is derivatization temperature, B is derivatization time, C is 4-MBA to nucleotide molar ratio, D is EDC to nucleotide molar ratio, E is NMM to nucleotide molar ratio 4-MBA);
FIG. 3 is a comparative chromatogram of the original nucleotides before and after derivatization (A is AMP, B is ADP, C is ATP, D is GMP, E is GDP, F is GTP, G is CMP, H is CDP, I is CTP, J is UMP, K is UDP, L is UTP) of the present invention;
FIG. 4 is a graph showing the comparison of LC-MS/MS detection effects before and after derivatization in the present invention (A is a graph showing the LC-MS/MS detection effects before chemical labeling, and B is a graph showing the LC-MS/MS detection effects after chemical labeling);
FIG. 5 is a chromatogram after derivatization of nucleotides with 4-MBA and 4-EBA, respectively, of the present invention (A is 4-EBA-ATP, 4-MBA-ATP, 4-EBA-ADP, 4-MBA-ADP, 4-EBA-AMP, 4-MBA-AMP chromatogram, B is 4-EBA-GTP, 4-MBA-GTP, 4-EBA-GDP, 4-MBA-GMP chromatogram, C is 4-EBA-CTP, 4-MBA-CTP, 4-EBA-CDP, 4-MBA-CDP, 4-EBA-CMP chromatogram, D is 4-EBA-UTP, 4-MBA-UTP, 4-EBA-UDP, 4-MBA-UMP, 4-UMP);
FIG. 6 is a graph showing comparison of nucleotide content in a DSS-modeled acute colitis model with that in a normal control group according to the present invention (A is AMP, B is GMP, C is CMP, D is UMP, E is ADP, F is GDP, G is CDP, H is UDP, I is ATP, J is GTP, K is CTP, L is UTP). P < 0.05, p < 0.01, p < 0.001;
fig. 7 is a graph showing comparison of nucleotide content in LPS-stimulated RAW264.7 cells with normal control (a is AMP, B is GMP, C is CMP, D is UMP, E is ADP, F is GDP, G is CDP, H is UDP, I is ATP, J is GTP, K is CTP, L is UTP) p < 0.01, p < 0.001, p < 0.0001 according to the present invention.
Detailed Description
The 4-methylbenzylamine in the present invention may be abbreviated as 4-MBA, and the 4-ethylbenzylamine may be abbreviated as 4-EBA,1- (3-
Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride can be abbreviated as EDC, N-methylmorpholine as NMM, electrospray ion source as ESI, multi-reaction monitoring as MRM, and liquid chromatography tandem mass spectrometry as LC-MS/MS.
Example 1
1. The volume ratio is 1:1 acetonitrile and water as solvents to prepare mother solutions with 10mM of each nucleotide standard substance;
2. preparing a working solution: (1) EDC, 4-MBA and NMM are sequentially dissolved in a mixed solution of acetonitrile and water in a volume ratio of 1:1 to prepare a 4-MBA derivatization solution, wherein the concentration of the 4-MBA solution is 62.5mM, the concentration of the EDC solution is 62.5mM and the concentration of the NMM solution is 12.5mM. (2) EDC, 4-EBA and NMM are sequentially dissolved in a mixed solution of acetonitrile and water in a volume ratio of 1:1 to prepare a 4-EBA derivative solution, wherein the concentration of the 4-EBA solution is 62.5mM, the concentration of the EDC solution is 62.5mM and the concentration of the NMM solution is 12.5mM.
3. Preparing a nucleotide mixed standard solution from the nucleotide mother solution in the step (1), mixing 20 mu L of nucleotide, adding 80 mu L of 4-EBA derivatization solution, uniformly mixing, reacting for 4 hours at 60 ℃, vibrating at 300rpm, and placing on ice for 2min after the reaction is finished to terminate the reaction, thereby obtaining the derivatization internal standard solution.
4. Detecting each standard curve to be detected by adopting an LC-MS/MS method, and establishing standard curves of various nucleotides by taking the nucleotide concentration as an abscissa and taking the peak area ratio of the 4-MBA marked nucleotide in the standard curve to be detected and the 4-EBA marked nucleotide in the corresponding derivatization internal standard solution as an ordinate.
5. The biological samples were protein precipitated by adding pre-chilled aqueous methanol (methanol: water=4:1, v/v), centrifuged at 13000rpm,4 ℃ for 10min, the supernatant removed and dried with nitrogen at room temperature.
6. Redissolving the biological sample treated in the step 5 into 20 mu L with the volume ratio of 1:1 acetonitrile and water, adding a 4-MBA derivatization reagent, uniformly mixing, reacting for 4 hours at 60 ℃, vibrating at 300rpm, placing on ice for 2 minutes after the reaction is finished to terminate the reaction, taking 45 mu L of biological sample reaction liquid into a 1.5mL centrifuge tube, adding 45 mu L of derivatization internal standard solution, adding 45 mu L of ultrapure water, swirling for 30sec, uniformly mixing, centrifuging at 13000rpm at 4 ℃ for 10 minutes, and taking supernatant for LC-MS/MS analysis.
The mass spectrometer used in the LC-MS/MS method was an 8045 mass spectrometer of shimadzu corporation (tokyo, japan) and was equipped with an electrospray ion source (ESI). The liquid chromatography portion was equipped with an LC-40B XR pump, CTO-40S column incubator, SIL-40C autosampler. Using a reverse phase chromatography column Shim-pack GIST C18 column (2.1X100 mm,2.0 μm); flow rate: 0.3mL/min; column temperature: 35 ℃; sample injection amount: 4. Mu.L; mobile phase: a is 5mM NH 4 HCO 3 And adjusting the pH to 7.8, B being pure acetonitrile; elution gradient: 0-3min,5% B;3-7min,5% -20% B;7-10min,20% -25% B;10-15min,25-30% B;15-20min,30% -60% B;20-22min,60% -80% B;22-24min,80% B;24-26min,80% -5% B; then equilibrated for 4min.
The mass spectrum conditions are as follows: adopting a positive ion mode, wherein the atomized air flow is 3L/min; the flow rate of the drying gas and the heating gas is 10L/min, the interface temperature is 250 ℃, the temperature of the DL tube is 250 ℃, and the temperature of the heating block is 400 ℃. Multiplex Reaction Monitoring (MRM) mode is used to detect post-derivatization nucleotides.
Example 2: analysis of colon tissue nucleotide in DSS-modeled acute colitis
C57BL/6J male mice (18-20 g) at 6-8 weeks of age, and after one week of adaptation, the mice were divided into two groups: the control group and the UC model group, respectively, were induced to UC by adding 3% (w/v) sodium dextran sulfate (DSS) to drinking water for 7 days, while the control group drinks pure water during this period. Mice were sacrificed on day 8 and colon tissue was removed. The collected sample should be handled as soon as possible to prevent degradation of the substance to be tested.
Taking 30mg of colon tissue in a 2mL centrifuge tube, adding 800 mu L of a mixed solution of precooled methanol and water with the water volume ratio of 4:1, putting the mixture into a grinder for grinding after adding magnetic beads, grinding for 10 times at intervals of 10sec every 60sec, centrifuging the homogenate at 13000rpm for 10min at 4 ℃, taking out supernatant, and drying with nitrogen at room temperature.
The dried sample was reconstituted with 20. Mu.L of acetonitrile in water (acetonitrile: water=1:1, v/v), 80. Mu.L of 4-MBA derivatization solution was added, after reaction, 45. Mu.L to 1.5mL of EP tube was taken, 45. Mu.L of derivatization internal standard solution was added, 45. Mu.L of pure water was added, vortexing was performed uniformly, and the mixture was centrifuged at 13000rpm at 4℃for 10min, and the supernatant was taken for LC-MS/MS analysis.
The standard curve of each nucleotide ranges from 5 to 500. Mu.M for AMP, ADP, ATP, from 0.5 to 50. Mu.M for CDP and CTP, and from 1 to 100. Mu.M for the remaining nucleotides. The comparison of the nucleotide contents measured is shown in FIG. 6.
Example 3: analysis of nucleotides in LPS stimulated RAW264.7 cells
Cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and at 37℃with 5% CO 2 Is divided into a control group and a Lipopolysaccharide (LPS) model group, the model group is stimulated with 200ng/mL LPS for 12h, and then the cells are collected.
Taking a certain amount of cultured cells, washing the cells with PBS buffer solution, placing the cells into a 1.5mL centrifuge tube, centrifuging the cells at 3000rpm and 4 ℃ for 5min, discarding the supernatant, adding 1mL of a mixed solution of precooled methanol and water with the volume ratio of 4:1, incubating the cells at 4 ℃ for 30min, and drying the obtained supernatant under nitrogen after vortex mixing, centrifuging the cells at 13000rpm and 4 ℃ for 10 min. The collected proteins were detected using a dioctanoic acid (BCA) protein detection kit according to the manufacturer's recommended protocol, and the cells were protein quantified.
The dried sample was reconstituted with 20. Mu.L of acetonitrile in water (acetonitrile: water=1:1, v/v), 80. Mu.L of 4-MBA derivatization solution was added, after reaction, 45. Mu.L to 1.5mL of EP tube was taken, 45. Mu.L of derivatization internal standard solution was added, 45. Mu.L of pure water was added, vortexing was performed uniformly, and the mixture was centrifuged at 13000rpm at 4℃for 10min, and the supernatant was taken for LC-MS/MS analysis.
The standard curves for each nucleotide ranged from AMP, ADP, UDP to 0.2. Mu.M, ATP, GTP from 2 to 200. Mu.M, GMP from 0.005 to 0.5. Mu.M, GDP from 0.02 to 2. Mu.M, CMP and CDP from 0.05 to 5. Mu.M, CTP from 0.5 to 50. Mu.M, UMP from 0.1 to 10. Mu.M, UDP from 0.2 to 20. Mu.M, UTP from 5 to 500. Mu.M, respectively. The comparison of the nucleotide contents measured is shown in FIG. 7.
The results show that in vitro studies, the level of the nucleotide in RAW264.7 cells was significantly reduced after LPS stimulation of the cells, and the differences were statistically significant. It was suggested that under inflammatory conditions, the cells released nucleotides from the inside of the cell to the outside of the cell for signaling. In vivo experiments, however, the change in GDP was most pronounced (p < 0.001), whereas CMP, UMP, CDP, UDP, GTP, etc. did not. The metabolism of UC and GDP and the mediated inflammatory signal reaction are most closely related, and the method is expected to provide a new clue and a new target point for preventing and treating ulcerative colitis.
In summary, inflammatory Bowel Diseases (IBDs), represented by Crohn's Disease (CD) and Ulcerative Colitis (UC), are chronic and palliative diseases that cause inflammation of the gastrointestinal tract. Prevalence is high in western developed countries, and up to 200 tens of thousands of people suffer from these diseases in europe [13] The incidence of IBD has been increasing in developing countries in south america, asia, africa and eastern europe [13,14] . Studies have shown that abnormal nucleotide metabolism is significantly associated with the development and progression of IBD. Under inflammatory stress conditions, nucleotides are released into the extracellular space through ubiquitin-binding proteins, gap junction half-channels (such as connexin 43), bind to specific purinergic receptors, and induce the occurrence of inflammatory responses. Different nucleotide-activated purinergic receptor subtypes differ. The P2X and P2Y11 receptors are mainly activated by extracellular ATP, the P2Y2 receptors are mainly activated by ATP and UTP, the P2Y1, P2Y12, P2Y13 receptors are mainly activated by ADP, and the P2Y12, P2Y13 receptors are mainly activated by ADP. P2Y4 is activated by UTP; P2Y6 and P2Y14 activation via UDP [15] . Therefore, analysis of the nucleotide in the tissue to find the abnormal nucleotide in UC will help to elucidate the pathological mechanism of UC and is also an urgent need for the discovery of new targets for treating UC.
In the early stages of the inflammatory response, macrophages first enter the affected area as effector cells, and then activate and recruit neutrophils, constituting the first line of defense of the body against infection. Thus, in examples 2 and 3, we used established methods to quantitatively analyze nucleotide levels in vivo and in vitro models of inflammation, with the aim of exploring nucleotide changes in the body after inflammation. In example 2, in vivo sample analysis, we model C57BL/6J mice using DSS, and the clinical manifestation and pathological features of this animal colitis are similar to human UC, which is the classical method for current study of UC pathogenesis and drug treatment. After modeling, colon tissues of the blank control group and the DSS model group were analyzed. In example 3, in vitro cell level analysis was performed. Research we use LPS to stimulate RAW264.7 cells for modeling, LPS is the main component of the cell wall of gram-negative bacillus, and can stimulate the cells to produce NO and TNF-alpha to form inflammatory response. Nucleotide quantitative analysis was performed on the cell lysates of the blank control group and the LPS-stimulated model group. As demonstrated by examples 2 and 3, the methods provided by this patent can be used for the determination of the nucleotide content in vivo and in vitro biological samples.
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Claims (7)
- 4-ethylbenzylamine 4-EBA and 4-methylbenzylamine 4-MBA are used as derivatization reagents for detecting nucleotide.
- 2. Use according to claim 1, characterized in that said use is 4-ethylbenzylamine 4-EBA and 4-methylbenzylamine 4-MBA as derivatizing reagents for detecting the free nucleotide content in a sample.
- 3. The use according to claim 2, wherein the sample is a cell or tissue.
- 4. Use according to claim 2 or 3, characterized in that the 4-methylbenzylamine 4-MBA derivatizes the free nucleotides in the sample, the 4-ethylbenzylamine 4-EBA derivatization standard nucleotides.
- 5. The use according to claim 4, wherein the volume ratio is 1: acetonitrile and water of 1 are used as solvents, EDC, 4-MBA and NMM are uniformly mixed to obtain 4-MBA derivatization solution; the volume ratio is 1: acetonitrile and water as solvent, EDC, 4-EBA and NMM are mixed uniformly to obtain 4-EBA derivatization solution.
- 6. A method for detecting a nucleotide, comprising the steps of:(1) The volume ratio is 1:1, acetonitrile and water are used as solvents to prepare a nucleotide standard substance solution;(2) The volume ratio is 1: acetonitrile and water as solvent, EDC, 4-EBA and NMM are added in sequence and mixed uniformly to obtain 4-EBA derivative solution;(3) Preparing a nucleotide mixed standard solution from the nucleotide mother solution obtained in the step (1), adding a 4-EBA derivatization solution, uniformly mixing, and reacting to obtain a derivatization internal standard solution;(4) The volume ratio is 1: acetonitrile and water as solvent, EDC, 4-MBA and NMM are added in sequence and evenly mixed to obtain 4-MBA derivatization solution;(5) Drawing a standard curve: selecting a nucleotide standard substance, diluting the nucleotide standard substance into a series of standard solutions with a certain concentration by using a solvent, adding the 4-MBA derivatization solution for derivatization reaction, adding the internal standard solution obtained in the step (3), and diluting to obtain solutions to be tested of all standard curves;(6) Detecting the solution to be detected of each standard curve by adopting an LC-MS/MS method, and establishing a standard curve of each nucleotide; wherein, the liquid chromatography conditions are as follows: chromatographic column: shimadzu Shim-pack GIST C18 column, 2.1X100 mm,2.0 μm; flow rate: 0.3mL/min; column temperature: 35 ℃; sample injection amount: 4. Mu.L; mobile phase: a is 5mM NH 4 HCO 3 And adjusting the pH to 7.8, B being pure acetonitrile; elution gradient: 0-3min,5% B;3-7min,5% -20% B;7-10min,20% -25% B;10-15min,25% -30% B;15-20min,30% -60% B;20-22min,60% -80% B;22-24min,80% B;24-26min,80% -5% B; then balancing for 4min;the mass spectrum conditions are as follows: adopting a positive ion mode, wherein the atomized air flow is 3L/min; the air flow rate of drying and heating is 10L/min, the interface temperature is 250 ℃, the DL tube temperature is 250 ℃, and the heating block temperature is 400 ℃; the multiplex reaction monitoring MRM mode is used for detecting the nucleotide after derivatization;(7) Extracting a biological sample and precipitating proteins, and drying the obtained supernatant with nitrogen to obtain a sample A;(8) Dissolving the sample A obtained in the step (7) in a volume ratio of 1:1, adding 4-MBA derivatization solution into acetonitrile and water solvent, uniformly mixing and reacting until the nucleotide reaction is completed, adding the derivatization internal standard solution obtained in the step (3), diluting with water, uniformly mixing, and centrifuging to obtain a supernatant sample B;(9) And (3) carrying out reversed-phase liquid chromatography-electrospray mass spectrometry on the supernatant sample B obtained in the step (8), and carrying out quantitative analysis on the nucleotide.
- 7. The method of claim 6, wherein the step of providing the first layer comprises,wherein in the step (4), the derivatization temperature is 60 ℃, the derivatization time is 4h, and the mol ratio of the nucleotide to the 4-MBA is 1:5000; the molar ratio of the nucleotide to EDC is 1:5000; the molar ratio of the nucleotide to NMM is 1:1000;the biological sample in the step (7) is a tissue or a cell,the deproteinization method of the tissue comprises the following steps: adding the tissue into a 2mL centrifuge tube, adding precooled methanol and a mixed solution with a water volume ratio of 4:1, grinding in a grinder for 60sec each time, grinding for 10 times at intervals of 10sec each time, centrifuging the homogenate at 13000rpm and at 4 ℃ for 10min after grinding, and taking out supernatant to obtain the tissue;cell deproteinization method: washing cells with PBS buffer solution, placing into a 1.5mL centrifuge tube, centrifuging at 3000rpm for 5min to precipitate cells, discarding supernatant, adding precooled mixed solution of methanol and water with water volume ratio of 4:1, incubating at 4deg.C for 30min, vortex mixing, centrifuging at 13000rpm at 4deg.C for 10min, and taking out supernatant to obtain the final product;in the step (8), in the nucleotide LC-MS/MS analysis method based on the pairing derivatization technology, the volume ratio of the biological sample A to the internal standard reaction solution to the water is 1:1:1.
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| CN117907491A (en) * | 2024-03-12 | 2024-04-19 | 中国人民解放军军事科学院军事医学研究院 | Double-derivatization technology-based abasic site LC-MS/MS analysis method |
| CN117907491B (en) * | 2024-03-12 | 2024-06-04 | 中国人民解放军军事科学院军事医学研究院 | Double-derivatization technology-based abasic site LC-MS/MS analysis method |
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| CN117907491A (en) * | 2024-03-12 | 2024-04-19 | 中国人民解放军军事科学院军事医学研究院 | Double-derivatization technology-based abasic site LC-MS/MS analysis method |
| CN117907491B (en) * | 2024-03-12 | 2024-06-04 | 中国人民解放军军事科学院军事医学研究院 | Double-derivatization technology-based abasic site LC-MS/MS analysis method |
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