Derivatization reagent and application thereof in method for identifying amino metabolites

Abstract

The invention relates to the field of cell analysis related to cytobiology, in particular to a derivatization reagent and application thereof in a method for identifying amino metabolites. The invention provides a novel derivatization reagent 3-bromoquinoline succinimide ester, the derivatization reagent has bromine atoms and quaternary ammonium groups with natural positive charges, so that the response of the labeled metabolite in mass spectrum can be improved, and by utilizing the characteristic of natural isotope distribution of the bromine atoms, the sensitivity of the metabolite in mass spectrum can be improved. The labeled metabolite can generate a pair of isotope peaks with fixed intensity ratio and fixed m / z difference in the mass spectrum, so that the identification of unknown metabolite is facilitated. The living cells labeled by the derivatization reagent can be used for mass spectrometry, especially for the field of single cell mass spectrometry, the response of metabolites in the direct sample injection process is improved, follow-up identification is facilitated, and the blank of domestic related technologies is filled.

Description

Technical Field

[0001] The invention relates to the field of cell analysis related to cell biology, and in particular to an active succinimidyl ester derivatization reagent for rapid labeling of amino compounds in living cells and an application thereof. Background Art

[0002] At present, most of the derivatization methods at the single-cell level involve long-term offline chemical derivatization reactions after cell disruption, or the use of micrometer-opening glass capillaries preloaded with derivatization reagents to extract cell contents and achieve chemical derivatization reactions of cell contents in glass capillaries. The analysis throughput of these methods is too low, and the only high-throughput analysis methods cannot provide high coverage of metabolite types. In addition, the shortest interval between the extraction of cell metabolites and subsequent analysis of these analysis methods is several minutes. At present, there is a lack of a method that can simultaneously achieve metabolite labeling and high-throughput single-cell analysis in living cells to avoid the loss of cell activity during analysis and the failure to meet the requirements of in situ analysis.

[0003] Patent CN202010288305.2 "Single Cell Mass Spectrometry Analysis Method" discloses a method for determining the position of unsaturated double bonds of lipids in single cells by mass spectrometry after derivatization. However, in the actual operation process, the cells need to be fixed with glutaraldehyde in advance, so when the subsequent mass spectrometry analysis is performed, the cells have been inactivated for too long, and the types and contents of metabolites will change significantly. This is not conducive to obtaining the true metabolic spectrum of the cell. Patent CN202211625959.5 "A Single Cell Online Chemical Derivatization Mass Spectrometry Analysis Device" provides a single cell online photochemical derivatization mass spectrometry analysis device. The device is in a microfluidic chip by mixing the flow path containing cells and the flow path containing derivatization reagents online, so that the metabolites of the cells are extracted and labeled by the derivatization reagent flow, thereby realizing the improvement of the response signal of the metabolites when they enter the mass spectrometry analysis later. However, in the actual operation process, the time that the cells are in contact with the derivatization reagent is too short, and the reaction efficiency of the derivatization reagent will not be very high, which makes the labeled metabolites fail to achieve the effect of offline reaction. In addition, high concentrations of derivatization reagents will also inhibit the mass spectrometry response of other metabolites. Patent CN111808750A "A microfluidic chip device and a method and application for in situ detection of living single cells" provides a microfluidic chip device and a method and application for in situ detection of living single cells. In the microfluidic chip, the fluid in the microchannel flows through the cell culture control unit for holding the target cells to extract the secretions of a single cell or its intracellular metabolites, followed by mass spectrometry analysis. Because the liquid in the flow path will cause severe dilution of the secretions and metabolites of a single cell, this method can only detect substances with high abundance. In addition, the device has a low throughput and can only analyze one cell in a short time, which is not conducive to statistical analysis of the data. Summary of the invention

[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 invention object, 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 a starting material, 3-bromopropylamine bromide is used as a nucleophilic reagent, and acetonitrile is used as a 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 in turn to prepare 3-bromoquinolylpropylamine 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 cause 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 scheme, 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 to 3 times.

[0013] In the above technical scheme, 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 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 freeze-dried 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 metabolite with the derivatization reagent described in claim 1;

[0017] (2) The labeled derivatized product is subjected to mass spectrometry analysis to obtain the characteristic peak of the bromine isotope in the primary mass spectrum 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;

[0018] (3) Collecting the secondary mass spectrometry information of the m / z of step (2), that is, obtaining fragments of the amino metabolite.

[0019] In the above technical solution, further, 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.

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

[0021] In the above technical solution, further, the amino metabolite reacts with a derivatization reagent to obtain a derivatization product; preferably, the adherent cells are cultured in a culture dish to a confluence of 60%-90%, the culture medium is removed, and after rinsing with PBS, a derivatization reagent PBS solution is added and incubated at 37°C.

[0022] In the above technical solution, further, 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.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The derivatization reagent of the present invention has low cytotoxicity, can improve the mass spectrometry response of the labeled metabolites, and is helpful for the identification of the metabolite structure. The derivatization reagent structure has a bromine atom, a quaternary ammonium group and a succinimide ester functional group. The bromine atom can make the derivative product form a pair of isotope characteristic peaks with a fixed m / z phase difference in a specific ratio in the primary mass spectrum, which assists in the identification of amino metabolites; 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 pass through the cell membrane to reach the cytoplasmic matrix and improve the mass spectrometry response of the derivative product. The succinimide ester can make the derivatization reagent react with the primary amine functional group of the amino metabolite, so as to realize the rapid labeling of amino metabolites in living cells under physiological conditions, and has the characteristics of simple operation and mild conditions.

[0025] According to the method of the present invention, after the adherent cells and the derivatization reagent are incubated in situ, the metabolites of a single cell can be aspirated by a microinjection needle through a micromanipulation platform for subsequent direct injection mass spectrometry analysis; the cells can also be made into a cell suspension of appropriate concentration, which can be arranged and sorted by a microfluidic chip and then analyzed one by one by entering the mass spectrometer. By screening out the characteristic peaks with bromine isotope ratios, potential characteristic peaks with amino groups can be preliminarily screened out, and then the structure of the potential characteristic peaks can be determined by secondary mass spectrometry, thereby completing the analysis of amino metabolites in a single living cell. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] Figure 2 This is a synthetic route for derivatization reagents.

[0028] Figure 3 is the reaction equation for the labeling of metabolites by the derivatization reagent used in the present invention.

[0029] Figure 4 Flowchart for single-cell mass spectrometry analysis.

[0030] Figure 5 Schematic diagram of isotope feature-assisted identification. Taking the derivatization of glycine (mass spectrum m / z is 76.0399) as an example, after being labeled with the derivatization reagent used in the present invention, 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.

[0031] Figure 6 Figure 2 is the secondary mass spectra of glycine and its derivatives. Figure a is the key fragment of the secondary mass spectrum of the glycine standard sample, and Figure b is the key fragment of the secondary mass spectrum of the derivative product. The fragment at 58 produced by the derivative product corresponds to the fragment peak of glycine.

[0032] Figure 7 The cell survival rates of cells after incubation with the derivatization reagent for different time periods, wherein the cell survival rate at 0 minute, i.e., when the cells were not incubated with the derivatization reagent, was used as a comparison. DETAILED DESCRIPTION

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

[0034] The present invention discloses a method for preparing a derivatization reagent for in situ derivatization of metabolites in living cells and its implementation route. Figure 1As shown, the derivatization reagent used in the present invention has a succinimide ester functional group, which can react quickly and efficiently with a primary amine group under mild conditions; 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 improve the response signal of the derivative product in the mass spectrometry; the bromine atom on the quinoline ring of the derivatization reagent will split the mass spectrometry signal of the derivative product due to the abundance of natural isotopes, generating m / z signals with two structures of bromine 79 and bromine 81, whose m / z difference is 1.9979±0.001, the intensity ratio is 0.9-1.1, and the m / z increase value is 290.0055 or 292.0034, which is helpful to screen out metabolites with primary amine groups from a huge number of unidentified characteristic peaks in metabolomics.

[0035] Example 1

[0036] Prepare the derivatization reagent as follows:

[0037] The first step is the preparation of 3-bromoquinolylpropylamine bromide (compound 1). 3-bromoquinoline (1.04 g, 5 mmol) and 3-bromopropylamine bromide (4.34 g, 20 mmol) were dissolved in 40 mL of acetonitrile and refluxed at 130°C for 3 days. After the reaction was completed, the reaction solution was filtered to obtain a yellow precipitate, which was then washed with acetonitrile and methanol twice to obtain a yellow solid 3-bromoquinolylpropylamine bromide 2 (1.71 g, 4 mmol, yield 80%). 1H NMR (400 MHz, DMSO-d6, ppm) δ8.12 (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] Step 2, preparation of 3-bromoquinolyl succinimide ester. Compound 1 (1.28 g, 3 mmol) and DSC (1.54 g, 6 mmol) were added to 20 mL DMSO, 0.5 mL TEA was added as an organic base, and the mixture was reacted 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 phases were water (containing 0.1% trifluoroacetic acid by volume) and acetonitrile (containing 0.1% trifluoroacetic acid by volume), respectively. A linear gradient was used: 2-4% of the aqueous phase increased to 15-18% of the aqueous phase, which took 30 min, and the effluent from 24-28 min was collected and vacuum-lyophilized to prepare a light yellow solid 3-bromoquinolyl succinimide ester (1.45 g, 2.5 mmol, yield 83.4%). 1H NMR (400 MHz, DMSO-d6, ppm) δ8.12 (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, measured value [M]+: 406.0399 and 408.0379.

[0039] Example 2

[0040] The derivatization reagent 3-bromoquinolyl succinimide ester prepared in Example 1 was dissolved in PBS to obtain a concentration of 10 mM for later use.

[0041] The glycine standard (mass spectrum m / z 76.0399) and the above derivatization reagent were shaken in PBS at room temperature for 10 minutes. The glycine standard before the reaction and the derivatization product after the reaction were diluted 100 times with 140mM ammonium formate solution and analyzed by mass spectrometry. The results are as follows: Figure 5As shown in the figure, after glycine is labeled, its 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, satisfying the condition of characteristic peaks containing bromine isotopes. By analyzing the mass spectrometry data through the program, it can be distinguished from a bunch of unidentified characteristic peaks. After deducting 290.0055 or 292.0034 from the m / z of its derivative product, it can be reduced to obtain the hydrogenation mass number of glycine 76.0399, which is consistent with the m / z of the standard sample. Subsequently, the secondary mass spectrum information with an m / z of 366.0452 is collected by tandem mass spectrometry to obtain a fragment with an m / z of 58. By comparing it with the secondary mass spectrum fragments of the standard sample, it can be confirmed that the characteristic peak of 366.0452 is generated after glycine is labeled with a derivatization reagent. For characteristic peaks of unknown m / z with the above characteristics, the real m / z obtained after the above operation can be identified by matching the primary mass spectrum database; or the secondary mass spectrum of the original m / z can be collected and searched and identified by fragment ions.

[0042] It can be seen from the identification that the method of the present invention, after labeling the amino metabolite with the derivatization reagent 3-bromoquinolyl succinimide ester, identifying the amino metabolite by primary mass spectrometry and secondary mass spectrometry, has accurate results.

[0043] Example 3

[0044] Hela cells were cultured in a culture dish with DMEM medium containing fetal bovine serum to a confluence of 80%, the medium was removed, the cells were rinsed twice with PBS to remove the residual medium, and then an appropriate volume of the above-mentioned 10mM concentration of the derivatization reagent solution was added to the culture dish, and the culture dish was placed at room temperature or in a 37 degree Celsius incubator for 5 minutes. After the incubation was completed, the derivatization reagent solution was removed and rinsed twice with a 140mM ammonium formate aqueous solution to remove the residual derivatization reagent. The subsequent injection method takes high-throughput single-cell metabolomics analysis as an example. After the cells were digested and delaminated with 0.25% trypsin EDTA, complete serum-containing medium was added to terminate the digestion, the supernatant was removed by centrifugation, and the cells were resuspended with 1mL of 140mM ammonium formate solution, and the cell concentration was obtained using an automatic cell counter. After the cells are diluted to a suitable concentration, the diluted cell suspension is injected into the microfluidic chip at a flow rate of 1 μL / min using a syringe pump, so that the cells are dispersed and arranged in an orderly manner and enter the induction pulse electrospray ion source, so that the cells are broken and metabolites are ionized, so as to be detected and collected by mass spectrometry. The characteristic peaks of the primary mass spectrum collected are screened out to meet the m / z difference of 1.9979±0.001 and the corresponding intensity ratio is 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 is obtained. After that, the m / z after deduction is matched with the primary mass spectrum database in the network database (such as HMDB), and the structure of the above characteristic peaks is preliminarily identified. Further, the secondary mass spectrum data of the above characteristic peaks are collected by tandem mass spectrometry, and the obtained secondary mass spectrum data are matched with the secondary mass spectrum database in the network database to further identify the specific structure of the characteristic peaks.

[0045] Figure 7 It is the cell survival rate after incubating the cells with the derivative reagent of the present invention for different time periods. It can be seen that the cell survival rate reaches 80% after incubating for 5 minutes, which can meet the needs of subsequent experiments.

[0046] For any technician familiar with the art, without departing from the scope of the technical solution of the present invention, the technical content disclosed above can be used to make many possible changes and modifications to the technical solution of the present invention, or modified into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention should still fall within the scope of protection of the technical solution of the present invention.

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 a starting material, 3-bromopropylamine bromide is used as a nucleophilic reagent, and acetonitrile is used as a 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 in turn to prepare 3-bromoquinolylpropylamine bromide (Compound 1); (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 cause 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 to 1:6; the reaction temperature is 110-130° C., the reaction time is 3 to 5 days; and the reaction is rinsed 2 to 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 12-24h; the mobile phase for liquid phase separation and purification is water and acetonitrile, the water contains 0.1-0.5% trifluoroacetic acid by volume, and the acetonitrile contains 0.1-0.5% trifluoroacetic acid by volume, the effluent is collected, and vacuum freeze-dried to prepare 3-bromoquinolyl succinimide ester.

5. Use of the derivatization reagent according to claim 1 in identifying amino metabolites.

6. A method for identifying amino metabolites, 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 spectrum 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 of 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, characterized in that The amino metabolite reacts with the derivatization reagent to obtain a derivatization product; preferably, 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, a derivatization reagent PBS solution is added, and the cells are 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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