A derivatizing reagent and its use in a method for identifying amino metabolites

Abstract

The present application relates to the field of cell analysis related to cell biology, in particular to a kind of derivatization reagent and its application in the method for identifying amino metabolites.The present application provides a new derivatization reagent 3-bromoquinoline succinimidyl ester, the derivatization reagent has bromine atom and quaternary ammonium group with natural positive charge, which can improve the response of labeled metabolites in mass spectrometry, and using the characteristics of natural isotopic distribution of bromine atom, the labeled metabolites can produce a pair of isotopic peaks with fixed intensity ratio and fixed m / z difference in mass spectrometry, thereby facilitating the identification of unknown metabolites.The living cells labeled by the derivatization reagent of the present application can be used for mass spectrometry, especially in the field of single-cell mass spectrometry, to improve the response of metabolites in direct injection process and facilitate subsequent identification, and make up for the blank of domestic related technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell analysis related to cell biology, in particular to a kind of active succinimidyl ester derivatization reagent for rapid labeling of amino compounds in living cells and its application. BACKGROUND

[0002] Currently, the derivatization method at single cell level is mostly long-time offline chemical derivatization reaction after cell disruption, or using micron opening glass capillary pre-loaded with derivatization reagent to extract cell contents, and realizing chemical derivatization reaction of cell contents in glass capillary. These methods have too low analysis throughput, and the only high-throughput analysis method cannot provide high-coverage metabolite species. In addition, the interval time required for extracting cell metabolites to subsequent analysis of these analysis methods needs at least several minutes. Currently, there is a lack of a method that can realize metabolite labeling in living cells and high-throughput single cell analysis at the same time, avoiding cell inactivation during analysis and achieving in-situ analysis requirements.

[0003] In patent CN202010288305.2 "Single cell mass spectrometry analysis method", a method for determining the position of unsaturated double bond in lipids in single cells after derivatization by mass spectrometry secondary is disclosed. However, in actual operation process, the cell needs to be fixed in advance by glutaraldehyde, so that the cell has been inactivated for a long time when subsequent mass spectrometry analysis is performed, and the types and contents of metabolites will change significantly. This is not conducive to obtaining the real metabolic profile of the cell. In patent CN202211625959.5 "Single cell online chemical derivatization mass spectrometry analysis device", a single cell online photochemical derivatization mass spectrometry analysis device is provided. The device mixes the flow path containing cells and the flow path containing derivatization reagent in the microfluidic chip, so that the metabolites of the cells are extracted and labeled by the flow of derivatization reagent, thereby improving the response signal of the metabolites when they are subsequently analyzed by mass spectrometry. However, in actual operation process, the contact time between the cell and the derivatization reagent is too short, and the reaction efficiency of the derivatization reagent is not high, which makes the labeled metabolites not reach the effect of offline reaction. In addition, high concentration of derivatization reagent will inhibit the mass spectrometry response of other metabolites. In patent CN111808750A "Microfluidic chip device and in-situ detection method for living single cells and application", a microfluidic chip device and in-situ detection method for living single cells and application are provided. In the microfluidic chip, the fluid in the microfluidic channel flows through the cell culture manipulation unit for containing target cells, realizing the extraction of single cell secretion or intracellular metabolites, and then performing mass spectrometry analysis. Because the liquid in the flow path will cause serious dilution of the secretion and metabolites of single cells, this method can only detect high-abundance substances, in addition, the device has low throughput, and only one cell can be analyzed in a short time, which is not conducive to statistical analysis of data. SUMMARY

[0004] In order to solve the problems existing in the prior art, the present invention provides a derivatization reagent and a method for identifying amino metabolites in living cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] The present invention discloses a derivatization reagent, the molecular structure of which is as follows:

[0007]

[0008] In the above technical solution, further, the preparation method of the derivatization reagent comprises the following steps:

[0009] (1) 3-bromoquinoline is used as the starting material, 3-bromopropylamine bromide is used as the nucleophilic reagent, and acetonitrile is used as the reaction solvent to undergo a nucleophilic substitution reaction. After the reaction is completed, the reaction solution is filtered to obtain a yellow precipitate, which is then washed with acetonitrile and methanol to prepare 3-bromoquinolinylpropylamine bromide (Compound 1).

[0010] (2) Compound 1 is used as a reaction raw material, N,N'-disuccinimidyl carbonate (DSC) is used as a succinimide ester donor, triethylamine (TEA) is used as an organic base, and dimethyl sulfoxide (DMSO) is used as a reaction solvent to undergo an esterification reaction; after the reaction is completed, the reaction solution is purified by semi-preparative liquid phase separation to prepare 3-bromoquinolyl succinimide ester;

[0011]

[0012] In the above technical solution, further, in the step (1), the molar ratio of 3-bromoquinoline to 3-bromopropylamine bromate is 1:3 to 1:6; the reaction temperature is 110-130° C., the reaction time is 3 to 5 days, and elution is performed 2-3 times.

[0013] In the above technical solution, further, in the step (2), the molar ratio of compound 1 to DSC is 1:1.5 to 1:2.5; the reaction temperature is 25-30°C, and the reaction time is 12-24h; the mobile phase for the liquid phase separation and purification is water, acetonitrile, water containing 0.1-0.5% volume of trifluoroacetic acid), acetonitrile containing 0.1-0.5% volume of trifluoroacetic acid, the effluent is collected and vacuum-lyophilized to prepare 3-bromoquinolyl succinimide ester.

[0014] The present invention also provides an application of a derivatization reagent in identifying amino metabolites.

[0015] The present invention also provides a method for identifying amino metabolites, the method comprising:

[0016] (1) labeling the amino metabolic product with the derivatization reagent of claim 1;

[0017] (2) obtaining a pair of isotopic characteristic peaks with a fixed m / z difference in the first mass spectrum data of the labeled derivatization product, and subtracting the m / z increase caused by the structure of the derivatization reagent to obtain the m / z of the amino metabolic product;

[0018] (3) collecting the secondary mass spectrum information of the m / z in step (2) to obtain the fragments of the amino metabolic product.

[0019] In the technical solution, further, the pair of characteristic peaks with the bromine isotope has a m / z difference of 1.9979±0.001, and the corresponding intensity ratio is 0.9-1.1.

[0020] In the technical solution, further, the m / z increase caused by the structure of the derivatization reagent is 290.0055 or 292.0034.

[0021] In the technical solution, further, the amino metabolic product reacts with the derivatization reagent to obtain a derivatization product; preferably, the adherent cells are cultured in a culture dish to 60%-90% confluence, the culture medium is removed, the cells are washed with PBS, and then the derivatization reagent PBS solution is added for incubation at 37℃.

[0022] In the technical solution, further, the m / z obtained in step (2) is matched with a first mass spectrum database to complete preliminary identification; and the secondary mass spectrum information obtained in step (3) is matched with a secondary mass spectrum database to identify whether the specific structure of the characteristic peak is the amino metabolic product.

[0023] Compared with the prior art, the beneficial effects of the present application are:

[0024] The derivatization reagent of the present application has low cytotoxicity, can improve the mass spectrum response of the labeled metabolite, and is helpful for the identification of the structure of the metabolite. The derivatization reagent has a bromine atom, a quaternary ammonium group and a succinimidyl ester functional group on its structure. The bromine atom can make the derivatization product form a pair of isotopic characteristic peaks with a fixed m / z difference in the first mass spectrum, which can assist in the identification of the amino metabolic product. The nitrogen atom on the quinoline ring of the derivatization reagent is connected to the carbon chain to form a quaternary ammonium group with a positive charge, which can make the derivatization reagent more easily penetrate the cell membrane to reach the cytoplasmic matrix and improve the mass spectrum response of the derivatization product. The succinimidyl ester can react with the primary amine functional group of the amino metabolic product to realize rapid labeling of the amino metabolic product in living cells under physiological conditions, and has the characteristics of simple operation and mild conditions.

[0025] The metabolites of the single cell can be taken by a microinjection needle through a micro-operation platform after the in-situ incubation of the adherent cells and the derivatization reagent, and then directly injected into a mass spectrometer for subsequent analysis. The cells can also be made into a cell suspension with a suitable concentration, and then arranged and sorted by a microfluidic chip, and then analyzed by a mass spectrometer one by one. By screening the characteristic peaks with bromine isotope ratios, potential characteristic peaks with amino groups can be preliminarily screened, and then the structure of the potential characteristic peaks can be determined by secondary mass spectrometry, so that the analysis of the amino metabolites in the single living cell can be completed. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The structure of the derivatization reagent used for the cells of the present application is shown in the figure, and the appropriate volume and concentration are selected according to the amount of the cells.

[0027] Figure 2 The synthesis route of the derivatization reagent is shown in the figure.

[0028] Figure 3 The reaction equation for the metabolites labeled by the derivatization reagent used in the present application is shown in the figure.

[0029] Figure 4 The flow chart of the single cell mass spectrometry is shown in the figure.

[0030] Figure 5 The schematic diagram of the isotope characteristic assisted identification is shown in the figure, taking the derivatization of glycine (m / z is 76.0399) as an example, after being labeled by the derivatization reagent used in the present application, the m / z changes from 76.0399 to 366.0452 and 368.0429, which is a pair of characteristic peaks with an intensity ratio of 1:0.97.

[0031] Figure 6 The secondary mass spectrum of glycine and its derivative product is shown in the figure, Fig. a is the key fragment of the secondary mass spectrum of the glycine standard sample, and Fig. b is the key fragment of the secondary mass spectrum of the derivative product, and the fragment at 58 generated by the derivative product corresponds to the fragment peak of glycine.

[0032] Figure 7 The cell survival rates of the cells after incubation with the derivatization reagent for different times are shown in the figure, and the cell survival rate at 0 minute, i.e., the cell survival rate without incubation with the derivatization reagent, is taken as a comparison. DETAILED DESCRIPTION

[0033] The present application will be further described below in conjunction with specific embodiments, but the present application is not limited in any way by the embodiments.

[0034] The embodiment of the present application discloses a preparation method of a derivatization reagent for in-situ derivatization of metabolites in living cells and an implementation route thereof. Figure 1As shown, the derivative reagent used in the present application has a succinimidyl ester functional group, which can rapidly and efficiently react with a primary amine group under mild conditions; the nitrogen atom on the quinoline ring of the derivative reagent is connected to a carbon chain to form a quaternary ammonium group with a positive charge, which can improve the response signal of the derivative product in mass spectrometry; the bromine atom on the quinoline ring of the derivative reagent will cause the mass spectrometry signal of the derivative product to split due to natural isotopic abundance, resulting in m / z signals with two structures of bromine 79 and bromine 81, with an m / z difference of 1.9979±0.001, an intensity ratio of 0.9-1.1, an m / z increase value of 290.0055 or 292.0034, thereby helping to screen metabolites with primary amine groups from the large number of unidentified characteristic peaks in metabolomics.

[0035] Example 1

[0036] The derivative reagent was prepared by the following method:

[0037] First step, preparation of 3-bromoquinolinylpropylamine bromide (compound 1). 3-bromoquinoline (1.04 g, 5 mmol), 3-bromopropylamine bromide (4.34 g, 20 mmol) were dissolved in 40 mL of acetonitrile, and the reaction was refluxed at 130°C for 3 days. After the reaction was completed, the reaction liquid was filtered to obtain a yellow precipitate, which was washed with acetonitrile and methanol twice in turn to obtain yellow solid 3-bromoquinolinylpropylamine bromide 2 (1.71 g, 4 mmol, yield 80%). 1H NMR (400 MHz, DMSO-d6, ppm) δ 8.12 (s, 3H), 5.36 (t, J = 6.4 Hz, 1H), 4.10 (s, 2H), 2.82 (s, 2H), 2.56 (m, 4H), 2.33 (s, 1H), 2.12 (d, J = 6.4 Hz, 2H), 2.05-2.01 (m, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C12H14BrN2): Theoretical value: 265.0335 and 267.0315, measured value [M+H]+: 265.0339 and 267.0317.

[0038] Second step, preparation of 3-bromoquinolinyl succinimidyl ester. Compound 1 (1.28 g, 3 mmol) and DSC (1.54 g, 6 mmol) were added into 20 mL DMSO, 0.5 mL TEA was added as organic base, and the reaction was carried out at 25 °C for 24 h. After the reaction was completed, the reaction solution was purified by semi-preparative liquid phase separation, and the mobile phase was water (containing 0.1% by volume of trifluoroacetic acid) and acetonitrile (containing 0.1% by volume of trifluoroacetic acid), respectively, using a linear gradient: 2-4% of the water phase increased to 15-18% of the water phase for 30 min, and the effluent was collected at 24-28 min. Vacuum freeze-drying yielded 3-bromoquinolinyl succinimidyl ester (1.45 g, 2.5 mmol, yield 83.4%) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6, ppm) δ 8.12 (s, 3H), 5.36 (t, J = 6.4 Hz, 1H), 4.10 (s, 2H), 2.82 (s, 2H), 2.56 (m, 4H), 2.33 (s, 1H), 2.12 (d, J = 6.4 Hz, 2H), 2.05-2.01 (m, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C17H17BrN3O4): Theoretical value: 406.0397 and 408.0377, Determination value [M]+: 406.0399 and 408.0379.

[0039] Example 2

[0040] The derivatization reagent 3-bromoquinolinyl succinimidyl ester prepared in Example 1 was dissolved in PBS to obtain a 10 mM concentration for standby use.

[0041] Glycine standard (mass spectrum m / z 76.0399) was reacted with the above-mentioned derivatization reagent in PBS at room temperature for 10 min. The glycine standard before reaction and the derivatization product after reaction were each diluted 100 times with 140 mM ammonium formate solution and then analyzed by mass spectrometry, and the results are shown in Table 1. Figure 5As shown, after the glycine is labeled, its m / z changes from 76.0399 to 366.0452 and 368.0429, which are a pair of characteristic peaks with an intensity ratio of 1:0.97, meeting the conditions of containing bromine isotope characteristic peaks. Through program analysis of mass spectrometry data, it can be distinguished from a pile of unidentified characteristic peaks. After the m / z of the derivative product is deducted by 290.0055 or 292.0034, the hydrogenated mass number 76.0399 of glycine can be obtained, which is consistent with the m / z of the standard sample. Then, the secondary mass spectrum information of m / z 366.0452 is collected by tandem mass spectrometry, and the fragment with m / z 58 is obtained, which can be confirmed by comparing the secondary mass spectrum fragments of the standard sample that the characteristic peak of 366.0452 is generated after the glycine is labeled by the derivative reagent. For unknown m / z characteristic peaks with the above characteristics, after the above operation, the true m / z obtained can be identified by matching the primary mass spectrometry database; or the secondary mass spectrum of the original m / z is collected, and the library identification is carried out through the fragment ions.

[0042] It can be seen through identification that the method for identifying amino metabolites after labeling the amino metabolites by the derivative reagent 3-bromoquinolyl succinimidyl ester according to the present application is accurate in result.

[0043] Example 3

[0044] Hela cells were cultured to 80% confluence in a culture dish with DMEM medium containing fetal bovine serum, the culture medium was removed, and the cells were rinsed twice with PBS to remove residual culture medium, then an appropriate volume of the above-mentioned 10 mM concentration of derivatization reagent solution was added to the culture dish, and the culture dish was incubated at room temperature or in a 37°C incubator for 5 minutes. After incubation, the derivatization reagent solution was removed, and the cells were rinsed twice with 140 mM ammonium formate aqueous solution to remove residual derivatization reagent. The subsequent sample injection method is illustrated by the example of high-throughput single-cell metabolomics analysis. After the cells were detached by 0.25% trypsin EDTA digestion, serum-containing complete medium was added to terminate the digestion, the supernatant was removed by centrifugation, the cells were resuspended with 1 mL of 140 mM ammonium formate solution, and the cell concentration was obtained using an automatic cell counter. Then the cells were diluted to an appropriate concentration, and the diluted cell suspension was injected into a microfluidic chip at a flow rate of 1 μL / min using a syringe pump, so that the cells were dispersed and arranged in order to enter the inductive pulse electro-spray ion source, so that the cells were broken and metabolites were ionized, thereby being detected and collected by mass spectrometry. The characteristic peaks of the collected primary mass spectrum were screened to obtain pairs of characteristic peaks that meet the conditions of m / z difference of 1.9979±0.001 and corresponding intensity ratio of 0.9-1.1. After deducting 290.0055 or 292.0034 from the m / z, the m / z of the corresponding substance before derivatization in the mass spectrum was obtained. Then the deducted m / z was matched with the primary mass spectrum database in the network database (such as HMDB) to preliminarily identify the structure of the above-mentioned characteristic peaks. Further, the above-mentioned characteristic peaks were collected by tandem mass spectrometry to obtain secondary mass spectrometry data, and the secondary mass spectrometry data was matched with the secondary mass spectrometry database in the network database to further identify the specific structure of the characteristic peaks.

[0045] Figure 7 is the cell survival rate after different incubation times of cells and the derivatization reagent of the present application. It can be seen that the survival rate of cells after 5 minutes of incubation reaches 80%, which can meet the needs of subsequent experiments.

[0046] For any person skilled in the art, many possible variations and modifications of the technical solutions of the present application can be made by using the above-mentioned disclosed technical content without departing from the scope of the technical solutions of the present application, or modified as equivalent examples of equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above-mentioned examples according to the technical essence of the present application, which does not deviate from the content of the technical solutions of the present application, should still belong to the scope of protection of the technical solutions of the present application.

Claims

1. A derivatization reagent, characterized in that: The molecular structure is as follows: 。 2. The derivatization reagent according to claim 1, characterized in that The preparation method of the derivatization reagent comprises the following steps: (1) 3-bromoquinoline is used as the starting material, 3-bromopropylamine bromide is used as the nucleophilic reagent, and acetonitrile is used as the reaction solvent to undergo a nucleophilic substitution reaction. After the reaction is completed, the reaction solution is filtered to obtain a yellow precipitate, which is then washed with acetonitrile and methanol to prepare 3-bromoquinolinylpropylamine bromide compound 1; (2) Compound 1 is used as a reaction raw material, N,N'-disuccinimidyl carbonate is used as a succinimide ester donor, triethylamine is used as an organic base, and dimethyl sulfoxide is used as a reaction solvent to undergo an esterification reaction; after the reaction is completed, the reaction solution is purified by semi-preparative liquid phase separation to prepare 3-bromoquinolyl succinimide ester; 。 3. The derivatization reagent according to claim 2, characterized in that In the step (1), the molar ratio of 3-bromoquinoline to 3-bromopropylamine bromate is 1:3-1:6; the reaction temperature is 110-130° C., the reaction time is 3-5 days, and the reaction is rinsed 2-3 times.

4. The derivatization reagent according to claim 2, characterized in that In the step (2), the molar ratio of compound 1 to DSC is 1:1.5 to 1:2.5; the reaction temperature is 25-30°C, and the reaction time is 12-24 h; the mobile phase for the liquid phase separation and purification is water and acetonitrile, with the water containing 0.1-0.5% by volume of trifluoroacetic acid and the acetonitrile containing 0.1-0.5% by volume of trifluoroacetic acid, and the effluent is collected and vacuum-lyophilized to prepare 3-bromoquinolyl succinimide ester.

5. Use of the derivatization reagent according to claim 1 in the preparation and identification of amino metabolite products.

6. A method for identifying amino metabolites not involving disease diagnosis or treatment, characterized in that The method comprises: (1) Labeling the amino metabolite with the derivatization reagent described in claim 1; (2) The labeled derivatized product is subjected to mass spectrometry analysis to obtain the characteristic peak of the bromine isotope in the primary mass spectrometry data, and the m / z increase caused by the structure of the derivatization reagent is deducted to obtain the m / z of the amino metabolite; (3) Collecting the secondary mass spectrometry information of the m / z in step (2), that is, obtaining fragments of the amino metabolite.

7. The method according to claim 6, characterized in that The characteristic peaks of bromine isotopes are paired characteristic peaks with an m / z difference of 1.9979±0.001 and a corresponding intensity ratio of 0.9-1.

1.

8. The method according to claim 6, characterized in that The m / z increase caused by the derivatization reagent structure is 290.0055 or 292.0034.

9. The method according to claim 6, wherein The amino metabolite reacts with the derivatization reagent to obtain a derivatized product; the adherent cells are cultured in a culture dish to a confluence of 60%-90%, the culture medium is removed, the cells are rinsed with PBS, and a derivatization reagent PBS solution is added and incubated at 37°C.

10. The method according to claim 6, characterized in that The m / z obtained in step (2) is matched with the primary mass spectrum database to complete preliminary identification; the secondary mass spectrum information obtained in step (3) is matched with the secondary mass spectrum database to identify whether the specific structure of the characteristic peak is an amino metabolite.

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