Method and kit for detecting redox activity metabolome by using matrix-assisted laser desorption ionization mass spectrometry

Abstract

The invention belongs to the technical field of mass spectrometry detection, and discloses a method and a kit for detecting redox activity metabolome by using matrix-assisted laser desorption ionization mass spectrometry. According to the method, 2, 4-dichloro-1-aminonaphthalene is taken as a matrix, and the redox activity metabolome is detected by using matrix-assisted laser desorption ionization mass spectrometry. According to the method, photoinduced electron transfer and redox potential difference are utilized, and electron gaining and losing, namely photoinduced electron soft ionization, are generated based on the redox potential difference, so that a sample loses or captures electrons, and corresponding cation free radicals, anion free radicals and fragment ions are generated; therefore, mass spectrometry and / or mass spectrometry imaging analysis of the redox activity metabolome can be realized.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry detection technology, specifically to a method and kit for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry. Background Technology

[0002] Cellular metabolism is the sum of a series of chemical reactions occurring within an organism, providing cells with energy, raw materials, and signaling molecules, and determining their physiological state and function. Redox metabolism is a crucial component of cellular metabolism, encompassing various redox reactions, post-redox modifications (PTMs), and a finely regulated network of cellular output. Redox-active metabolites can regulate cellular function by modulating the activity of metabolic enzymes and transcription factors, as well as by regulating gene expression and epigenetic modifications.

[0003] Mass spectrometry is currently the primary analytical tool for identifying the structure of metabolites. It mainly relies on the protonation-deprotonation mechanism, mixing the sample with an organic acid or base matrix and then bombarding it with a laser to convert various neutral molecules into charged ions for mass spectrometry analysis. Therefore, it is suitable for organic acids or bases with active hydrogen atoms. However, metabolites have various structures, and these substances have different net charges under physiological conditions. Examples include uncharged dipolar ions, polybasic acid anions with multiple charges, various cations, and neutral molecules. Many molecules cannot be protonated or deprotonated, making them difficult to detect using conventional mass spectrometry methods. Summary of the Invention

[0004] To address the problems existing in the background art, this invention provides a method and kit for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry. This invention uses 2,4-dichloro-1-aminonaphthalene, which has both photoactive and redox activity, as a matrix. By utilizing photoinduced electron transfer and redox potential difference, electron gain and loss occur based on the redox potential difference, i.e., photoinduced soft ionization of electrons, causing the sample to lose or capture electrons, generating corresponding cationic free radicals, anionic free radicals, and fragment ions, thereby enabling mass spectrometry analysis and / or mass spectrometry imaging analysis of the redox metabolomics. The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides the application of 2,4-dichloro-1-aminonaphthalene as a matrix for matrix-assisted laser desorption / ionization mass spectrometry detection.

[0005] According to the above scheme, 2,4-dichloro-1-aminonaphthalene is used as a matrix for mass spectrometry analysis and / or mass spectrometry imaging analysis of redox metabolites in biological tissue samples, body fluids or blood samples.

[0006] 2,4-Dichloro-1-aminonaphthalene possesses both photoactivity and redox activity. It contains a naphthalene ring, a functional group that absorbs specific wavelengths of laser light, but lacks active hydrogen, thus it cannot donate or accept protons, and therefore cannot protonate or deprotonate samples. 2,4-Dichloro-1-aminonaphthalene absorbs specific wavelengths of laser light, undergoing photoinduced electron transfer by losing or gaining electrons under irradiation, rather than protonation or deprotonation. Therefore, it can be used as a matrix for matrix-assisted laser desorption / ionization mass spectrometry (LASIK) detection of redox-active substances.

[0007] Secondly, the present invention provides a method for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry, using 2,4-dichloro-1-aminonaphthalene as a matrix, and using matrix-assisted laser desorption / ionization mass spectrometry to detect redox-active metabolomics.

[0008] According to the above scheme, the wavelength of the laser used is 210-370 nm.

[0009] According to the above scheme, the wavelength of the laser used is 266nm or 355nm.

[0010] High-frequency pulsed lasers are used, with a pulse width of 3 ns - 5 ns and a pulse frequency of 1000 Hz ~ 10000 Hz.

[0011] According to the above scheme, the method for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry includes the following steps: S1. Dissolve 2,4-dichloro-1-aminonaphthalene in a solvent to prepare a matrix solution; S2. Prepare a liquid or solid powder sample of the metabolite by mixing the matrix solution with the liquid or solid powder sample of the metabolite, spotting the mixture onto a sample target, and drying; or Prepare tissue section samples by spraying the matrix solution onto the surface of the tissue sections and then drying. S3. Place the processed sample into the mass spectrometer and perform mass spectrometry detection in either positive or negative ion mode.

[0012] According to the above scheme, in step S1, the concentration of the matrix in the matrix solution is 0.02 mol·L⁻¹. -1 -0.05 mol·L -1 The solvent is an aqueous solution of acetonitrile or an alcoholic solution of acetonitrile with a concentration of 50%-60%, or other solvents that can dissolve the matrix and the sample.

[0013] According to the above scheme, in step S3, the choice between positive ion mode and negative ion mode depends on the redox potential difference between the matrix and the sample molecules.

[0014] According to the above scheme, in step S2, the preparation steps for liquid or solid powder samples are as follows: After washing the biological cell sample and removing the culture medium, cell lysis buffer is added, and the cell walls are disrupted by ultrasound. Metabolite extraction reagent is then added to obtain a liquid sample. Optionally, the liquid sample is concentrated by centrifugation, organic solvent is removed, and then freeze-dried to obtain a solid powder sample. The liquid or solid powder sample is mixed with the matrix solution, and the mixing ratio is determined by the sample content, which can be in the range of 1:1 to 1:10.

[0015] According to the above scheme, in step S2, the preparation steps of the tissue sample sections are as follows: Using a cryostat, thin sections of 5-20 micrometers are prepared from animal and plant tissues onto a conductive glass slide. The prepared tissue sections are then placed in a matrix sprayer, and a matrix solution is uniformly sprayed onto the surface of the tissue sections at a set temperature and nitrogen flow rate. Preferably, the conductive glass slide is an indium tin oxide (ITO) glass slide.

[0016] For liquid or solid powder samples, mass spectrometry analysis is performed to search the mass spectrometry database of metabolites and identify redox-active metabolites. For tissue section samples, mass spectrometry analysis and image reconstruction are performed, and the mass spectrometry database of metabolites is searched based on the mass spectrometry imaging analysis data to identify redox-active metabolites.

[0017] 2,4-Dichloro-1-aminonaphthalene is a photoactive and redox-active organic matrix. It is co-crystallized by mixing with liquid or solid powder samples or spraying onto the surface of tissue sections. Upon irradiation with a laser of a specific wavelength, matrix molecules gain or lose electrons based on the redox potential difference, while sample molecules capture or lose electrons, generating anionic or cationic free radicals. This leads to the breaking of chemical bonds caused by unpaired electrons, producing specific fragment ions. By detecting these anionic, cationic, and fragment ions, the redox-active metabolic components in the sample can be analyzed. For tissue sections, their spatial distribution can be further imaged.

[0018] This method involves co-crystallizing a redox-active metabolite with an organic small molecule matrix, 2,4-dichloro-1-aminonaphthalene, followed by electron transfer under laser bombardment. The generated ions are then identified using a mass spectrometer in either positive or negative ion mode. This method is suitable for mass spectrometry analysis and / or mass spectrometry imaging analysis of redox-active metabolomics in biological tissues, body fluids, or blood samples.

[0019] Thirdly, the present invention provides a kit for detecting redox-active metabolomics based on matrix-assisted laser desorption / ionization mass spectrometry, comprising 2,4-dichloro-1-aminonaphthalene.

[0020] The beneficial effects of this invention are: Compared with existing matrix-assisted laser desorption / ionization mass spectrometry analysis based on organic small molecule acids or bases, this invention uses 2,4-dichloro-1-aminonaphthalene as the matrix. The sample does not undergo protonation or deprotonation, but rather electron gain and loss occurs based on its redox potential difference, i.e., photoinduced soft electron ionization. The sample loses electrons to generate cationic free radicals and fragment ions, or captures electrons to generate anionic free radicals and fragment ions. The mass spectrometer detects redox active metabolites with different structures and obtains spatial distribution imaging. This method is applicable to the detection of substances that cannot be protonated or deprotonated, and is especially suitable for the detection of redox-active metabolomics in tissue, body fluid or blood samples; The method of the present invention is simple and easy to operate. It only requires mixing the sample with the matrix or spraying the matrix onto the surface of the tissue section. The quality is stable and there is no need to transfer the sample in the middle. Attached Figure Description

[0021] Figure 1 This is the mass spectrum of 2,4-dichloro-1-aminonaphthalene obtained in positive ion mode in Example 1 of the present invention; Figure 2 This is the mass spectrum of 9-aminoacridine obtained in positive ion mode in Example 1 of the present invention; Figure 3 This is an image of the co-crystallization formed between 2,4-dichloro-1-aminonaphthalene and the sample in Example 2 of the present invention; Figure 4 This is a mass spectrometry imaging analysis image obtained using matrix 2,4-dichloro-1-aminonaphthalene in Example 3 of the present invention. The sample is a breast cancer tissue section. The image shows the spatial distribution of ion m / z 62.9635 in the tumor area and adjacent normal area. Figure 5 This is a mass spectrometry imaging analysis image obtained using matrix 2,4-dichloro-1-aminonaphthalene in Example 4 of the present invention. The sample is a breast cancer tissue section. The image shows the spatial distribution of ion m / z 74.0027 in the tumor area and adjacent normal area. Its color scale is the same as... Figure 4 ; Figure 6 The images shown are mass spectrometry images obtained in Example 5 of this invention using the photoactive matrix 2,4-dichloro-1-aminonaphthalene and the common basic matrix 9-aminoacridine. The sample is a breast cancer tissue section. The images show the spatial distribution of ions m / z 133.0129 (malic acid) and m / z 89.0236 (lactic acid) in the tumor area and adjacent normal area. The color scale is the same as... Figure 4 . Detailed Implementation

[0022] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] Through exploratory research, the inventors discovered that 2,4-dichloro-1-aminonaphthalene possesses both photoactivity and redox activity. It contains a naphthalene ring, a functional group capable of absorbing specific wavelengths of laser light, but lacks active hydrogen. It can absorb specific wavelengths of laser light, and under laser irradiation, it does not undergo protonation or deprotonation, but rather loses or gains electrons, i.e., it undergoes photoinduced electron transfer. Therefore, it can be used as a matrix for laser desorption / ionization mass spectrometry (LAS-MS) detection of redox-active substances, especially for mass spectrometry analysis and / or mass spectrometry imaging analysis of redox-active metabolomics substances.

[0024] 4-Dichloro-1-aminonaphthalene can absorb laser light in the wavelength range of 210-370 nm and undergo redox reactions under laser irradiation within this range, losing or gaining electrons and undergoing photoinduced electron transfer with the sample. This is used for matrix-assisted laser desorption / ionization mass spectrometry (MAMS) to detect substances with redox activity. Specifically, the wavelength can be 266 nm, 355 nm, etc. In some specific embodiments of this invention, a laser wavelength of 355 nm is used.

[0025] Example 12, Mass spectrum of 4-dichloro-1-aminonaphthalene The mass spectrum of 2,4-dichloro-1-aminonaphthalene was determined by means of a matrix-assisted laser desorption / ionization quadrupole-time-of-flight tandem mass spectrometer in positive ion mode, by bombarding 2,4-dichloro-1-aminonaphthalene with a 355 nm laser.

[0026] The mass spectrum of 2,4-dichloro-1-aminonaphthalene is shown below. Figure 1 As shown, located m / z The ion at 210.9944 is a cationic radical formed after the matrix 2,4-dichloro-1-aminonaphthalene loses electrons; the protonated product was not detected. m / z The products of the three ionic radical coupling reactions at positions 315, 350, and 386, among which... m / z The ion at position 386 is the product of two matrix couplings losing one HCl ion, and then losing another HCl ion. m / z 350 ions, further removing Cl to become m / z 315 ions. This demonstrates that the photoactive matrix 2,4-dichloro-1-aminonaphthalene can lose electrons to become a cationic radical, rather than a cation.

[0027] 9-Aminoacridine is a small molecule base that is often used as a matrix for laser desorption / ionization mass spectrometry. The specific method for determining the mass spectrum of 9-aminoacridine is as follows: the 9-aminoacridine solution is transferred to the sample target using a pipette, and after the reagent has evaporated naturally, the sample target is placed in the mass spectrometer for measurement.

[0028] The mass spectrum of 9-aminoacridine is shown below. Figure 2 As shown, located m / z The ion at 195.0961 is a positively charged ion formed after the protonation of the matrix 9-aminoacridine. No electron-losing cationic radical was detected. This indicates that 9-aminoacridine cannot lose electrons, but instead gains a proton to become a cation. This is a completely different mechanism of action from 2,4-dichloro-1-aminonaphthalene. No proton-gaining ion peak was detected in 2,4-dichloro-1-aminonaphthalene under laser bombardment; instead, a cationic radical formed after gaining and losing electrons was detected. This proves that 2,4-dichloro-1-aminonaphthalene does not gain or lose protons, but rather gains and loses electrons.

[0029] Example 2 The 2,4-dichloro-1-aminonaphthalene matrix was mixed with the amino acid standard glutamine and dried to obtain a co-crystallization. The specific method is as follows: a 0.05 mol·L⁻¹ aminonaphthalene solution was prepared... -1 ) and glutamine solution (0.01 mol·L -1 Mix in a ratio of 1:1 to 1:10, transfer to a glass slide using a pipette, allow to air dry naturally, and then image under an optical microscope.

[0030] Optical microscopy imaging results of the co-crystallization of 2,4-dichloro-1-aminonaphthalene matrix and amino acid standard glutamine are shown below. Figure 3 As shown, 2,4-dichloro-1-aminonaphthalene can co-crystallize well with the sample, and the matrix can well encapsulate the sample. In the subsequent assisted laser desorption / ionization mass spectrometry process, the laser can avoid directly burning the sample. At the same time, the matrix absorbs the laser energy and transfers the energy to the sample molecules, while electron transfer occurs between the matrix and the sample.

[0031] Example 3: Mass spectrometry imaging analysis of small molecule metabolites in human breast cancer tissue sections The method of this invention is used to detect the distribution of metabolites capable of electron gain and loss in human breast cancer tissue sections and to acquire their mass spectrometry images. The specific operation steps are as follows: 1) Place human breast cancer tissue in the sample holder, apply tissue freezing embedding agent to the part that directly contacts the sample holder, and place it in the -40°C freezing chamber of the cryostat.

[0032] 2) Slice the sample obtained in step 1) to obtain thin slices of 5-20 micrometers on a conductive glass slide.

[0033] 3) Spray the tissue sections obtained in step 2) with a 2,4-dichloro-1-aminonaphthalene matrix.

[0034] 4) Place the slice obtained in step 3) into a mass spectrometer, bombard the sample with a 355 nm laser in negative ion mode, and collect data.

[0035] 5) Reconstruct the image from the data obtained in step 4) to obtain a mass spectrum image at m / z 62.9635.

[0036] The ion located at m / z 62.9635 is an anionic radical that carries a negative charge after capturing an electron, and its mass spectrum image is as follows. Figure 4 As shown, this ion is located in the tumor area.

[0037] A small molecule base, 9-aminoacridine, was used as the matrix for laser desorption / ionization mass spectrometry (LASMS). The difference lay in the matrix used in step 4), while all other conditions remained the same. No significant signal was detected at m / z 62.9635. This is because ordinary matrices require sample molecules to have active protons, meaning they can be protonated or deprotonated. In negative ion mode, the sample molecules could not be detected with ordinary matrices, indicating that they could not be deprotonated. However, they could be detected using 2,4-dichloro-1-aminonaphthalene as the matrix, indicating that this molecule has redox activity and can capture electrons.

[0038] Example 4: Mass spectrometry imaging analysis of small molecule metabolites in human breast cancer tissue sections The method of this invention is used to detect the distribution of metabolites capable of electron gain and loss in human breast cancer tissue sections and to acquire their mass spectrometry images. The specific operation steps are as follows: 1) Place human breast cancer tissue in the sample holder, apply tissue freezing embedding agent to the part that directly contacts the sample holder, and place it in the -40°C freezing chamber of the cryostat.

[0039] 2) Slice the sample obtained in step 1) to obtain thin slices of 5-20 micrometers on a conductive glass slide.

[0040] 3) Spray the tissue sections obtained in step 2) with a 2,4-dichloro-1-aminonaphthalene matrix.

[0041] 4) Place the slice obtained in step 3) into a mass spectrometer, bombard the sample with a 355nm pulsed laser in negative ion mode at a pulse frequency of 10000 Hz, and collect data.

[0042] 5) Reconstruct the image from the data obtained in step 4) to obtain a mass spectrometry image at m / z 74.0275.

[0043] The ion located at m / z 74.0275 is an anionic radical that carries a negative charge after capturing an electron, and its mass spectrum image is as follows. Figure 5 As shown, this ion is located in the tumor area.

[0044] The small molecule base 9-aminoacridine was used as the matrix for laser desorption / ionization mass spectrometry detection. The difference was that the matrix used in step 4) was different, but all other conditions were the same. No obvious signal was detected at m / z 74.0275.

[0045] Example 5 The method of this invention is used to image human breast cancer tissue sections and analyze the distribution of organic acids in the tissue sections. This invention uses malic acid and lactic acid as examples, employing 2,4-dichloro-1-aminonaphthalene and 9-aminoacridine (9-AA) as malic acid ... m / z (133.0129) and lactic acid ( m / z The distribution of 89.0236 g in tissue sections was determined. Measurement conditions were: negative ion mode, laser wavelength 355 nm. Results are as follows: Figure 6 As shown, when 9-AA is used as the matrix, stronger absolute intensity signals are generated in the detection of malic acid and lactic acid, but the spatial resolution is low and it cannot distinguish between tumor areas and adjacent tissues. When 2,4-dichloro-1-aminonaphthalene is used as the matrix, the absolute intensity of malic acid and lactic acid signals is lower than that of the control, but the spatial resolution is high and it can distinguish between tumor areas and adjacent tissues. Analysis of the above results: 9-AA has a pKa of 9.99, making it a moderately strong base. It can completely deprotonate lactic acid and malic acid, producing strong absolute intensity signals in both the tumor area and adjacent tissues. Therefore, it cannot distinguish between the tumor area and adjacent tissues, exhibiting a leveling effect. However, 2,4-dichloro-1-aminonaphthalene is not basic and cannot deprotonate these organic acids. Only unionized neutral molecules can capture electrons. The tumor microenvironment is acidic, and in an acidic environment, the ionization of organic acids is inhibited. Therefore, the acidic tumor area contains more neutral unionized organic acids. Thus, 2,4-dichloro-1-aminonaphthalene exhibits a distinguishing effect between the tumor area and adjacent tissues, effectively differentiating between the tumor area and adjacent tissues. Therefore, although 9-AA can produce a stronger absolute intensity signal, it cannot effectively distinguish between the tumor area and adjacent tissues.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of 2,4-dichloro-1-aminonaphthalene as a matrix for matrix-assisted laser desorption / ionization mass spectrometry detection.

2. The application according to claim 1, characterized in that, Mass spectrometry and / or mass spectrometry imaging analysis of redox-active metabolomics substances in biological tissue samples, body fluids, or blood samples.

3. A method for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry, characterized in that, Using 2,4-dichloro-1-aminonaphthalene as a matrix, matrix-assisted laser desorption / ionization mass spectrometry was used to detect redox-active metabolomes.

4. The method for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry according to claim 3, characterized in that, The wavelength of the laser used is 210 nm-370 nm.

5. The method for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry according to claim 4, characterized in that, The wavelength of the laser used is 266nm or 355nm.

6. The method for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry according to any one of claims 3 to 5, characterized in that, The steps include: S1. Dissolve 2,4-dichloro-1-aminonaphthalene in a solvent to prepare a matrix solution; S2. Prepare a liquid or solid powder sample of the metabolite by mixing the matrix solution with the liquid or solid powder sample of the metabolite, spotting the mixture onto a sample target, and drying; or Prepare tissue section samples by spraying the matrix solution onto the surface of the tissue sections and then drying. S3. Place the processed sample into the mass spectrometer and perform mass spectrometry detection in either positive or negative ion mode.

7. The method for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry according to claim 6, characterized in that, In step S1, the concentration of the matrix in the matrix solution is 0.02 mol·L⁻¹. -1 - 0.05 mol·L -1 The solvent is an aqueous solution of acetonitrile or an alcoholic solution of acetonitrile with a concentration of 50%-60%, or other solvents that can dissolve the matrix and the sample.

8. The method for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry according to claim 7, characterized in that, In step S2, the liquid sample preparation steps are as follows: after washing the biological cell sample and removing the culture medium, cell lysis buffer is added, and the cell walls are broken by ultrasound. Metabolite extraction reagent is added to obtain the liquid sample. The preparation steps for the solid powder sample are as follows: the liquid sample is concentrated by centrifugation, the organic solvent is removed, and then the solid powder sample is obtained by freeze drying. The steps for preparing tissue sample sections are as follows: animal and plant tissues are prepared into thin sections of 5-20 micrometers on conductive glass slides using a cryostat.

9. The method for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry according to claim 6, characterized in that, In step S3, the choice between positive ion mode and negative ion mode depends on the redox potential difference between the matrix and sample molecules.

10. A kit for detecting redox-active metabolomics using matrix-assisted laser desorption / ionization mass spectrometry, characterized in that, Including 2,4-dichloro-1-aminonaphthalene.

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