A method for mass spectrometry imaging detection of thiol compounds in a tissue

Abstract

The application discloses a mass spectrometry imaging detection method and application of a mercapto compound, and belongs to the technical field of mass spectrometry detection. The method at least comprises the following steps: synthesizing a derivatization reagent, preparing a derivatization system, spraying the derivatization system solution to the surface of a prepared tissue section, performing matrix spraying on the tissue section after derivatization, and performing MALDI mass spectrometry imaging analysis. The application can effectively improve the detection sensitivity of endogenous and exogenous mercapto compounds, realizes visual analysis of the mercapto compounds in biological tissues, and therefore has good practical popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of mass spectrometry detection technology, specifically relating to the development and application of a chemical derivatization and mass spectrometry imaging method for a thiol compound. Background Technology

[0002] Glutathione (GSH) is an important antioxidant, abundantly found in the cytosol and synthesized intracellularly in a tightly regulated manner. It possesses various physiological functions, including protection against reactive oxygen species and nitrogen, antioxidant defense, and maintenance of cellular thiol status. Due to its high oxygen consumption, the human brain readily produces reactive oxygen species. GSH plays a crucial role in the brain's antioxidant defense and maintenance of redox homeostasis.

[0003] A major challenge in analyzing thiol-containing compounds (such as GSH and cysteine) is their instability; therefore, stabilizing these compounds during sample preparation to prevent oxidation and interference with detection is crucial. Derivatization combined with LC-MS analysis is a common strategy for thiol detection. Over the past few decades, various methods have been developed for the derivatization detection of thiol compounds, primarily falling into two categories: reactive halogens and maleimides. While these methods should be widely applied to the detection of thiol compounds, limitations in sample preparation, tissue / organ heterogeneity, and loss of spatial information hinder the acquisition of more refined sample information. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) is an emerging bioanalytical tool that allows for direct spatially resolved tissue analysis and mapping of the spatial distribution of molecules of interest within tissues. Considering the limited detection sensitivity of low molecular weight compounds in MALDI-MSI, the histochemical derivatization (OTCD) technique developed using derivatization combined with MALDI-MSI has been used to achieve the detection of low-abundance compounds containing functional groups such as amino, carboxyl, carbonyl, thiols, and double bonds. OTCD has proven to be an important approach to addressing these issues and is increasingly being used in MALDI-MSI studies.

[0004] Over the past few decades, thiol-based chemical derivatization has seen significant advancements in LC-MS detection, but its application in mass spectrometry imaging remains limited. Fulop et al. designed and synthesized a derivative reagent, (E)-2-cyano-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-(4-hydroxyphenyl)acrylamide (CHC-Mal), for the selective detection of free thiols in mass spectrometry imaging. Although reported methods have detected small thiol-containing molecules, the products are mostly in the [M+CHC+K]+ form, and the derivatization mass spectrometric response of small molecule compounds is not directly obtained. Furthermore, derivatization requires complex pretreatment procedures, which may lead to spatial shifts of the analyte molecule during slicing. Therefore, the development of rapid and concise mass spectrometry imaging methods is crucial for the detection of thiol-containing compounds. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a mass spectrometry imaging detection method for thiol compounds in tissues. This invention synthesizes a derivatization reagent that can be applied to in-situ derivatization of thiol compounds for MALDI mass spectrometry imaging, significantly improving the detection sensitivity of thiol compounds in tissues and enabling the visual analysis of endogenous metabolites containing thiol groups in biological tissues. The derivatization reagent is synthesized by reacting the oxonium ions of a pyranonium salt with the primary amine contained in an N-substituted maleimide (containing an amino group). After separation and purification, a high-purity derivatization product is obtained. A derivatization solution system is prepared and sprayed onto the surface of tissue sections for mass spectrometry imaging detection of thiol compounds. Our proposed method has been successfully applied to the MSI of four thiol compounds, including GSH, in mouse brains. This method has also been successfully applied to mass spectrometry imaging of thiol compounds in the brains of CSDS model mice, elucidating the changing trends of thiol compounds in different brain regions.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This application provides a method for the derivatization of thiol compounds, comprising at least:

[0008] A mixture containing a thiol compound and a derivatizing reagent is subjected to a derivatization reaction to obtain the derivatized product;

[0009] The derivatizing reagent is compound I.

[0010]

[0011] Optionally, R1-R5 can be hydrogen or one or more of the following: methyl, ethyl, phenyl, alkyl, hydroxyl, alkoxy, or halogen (one or more of F, Cl, Br, I), with the number of substituents being 1-5, preferably 1-2. The number of substituents can be 0, one, two, three, four, or five; the length of the alkyl chain is 5 ≥ n ≥ 0.

[0012] The reaction solvent for the pyranium salt and N-substituted maleimide (containing amino group) is an organic reagent, which may be one or a combination of two or more of DCM, ACN, MeOH, THF, DMF and chloroform.

[0013] The acid-base catalyst is a base catalyst and an acid catalyst. Specifically, the reaction involves adding a base first, followed by the addition of an acid to continue the reaction. The base catalyst is one or two of triethylamine or N,N-diisopropylethylamine, and the acid catalyst is one, two, or three of formic acid, acetic acid, or benzoic acid. The molar ratio of the acid-base catalyst to the pyranium salt is 1:10-10:1 (molar ratio of acid to base to pyranium salt), preferably 0.5:1-2 (molar ratio of acid to base to pyranium salt), more preferably 1:1 (molar ratio of base to pyranium salt) or 2:1 (molar ratio of acid to pyranium salt). The derivatization system is prepared by mixing the derivatization reagent, acetic acid, and triethylamine, and directly spraying the mixture onto the surface of the tissue section without incubation, allowing it to air dry naturally.

[0014] In the derivatization system, the concentration of the derivatizing reagent is 0.05-5 mM (preferably 0.1-1 mM, more preferably 0.1-0.5 mM); the concentration of the catalyst TEA (triethylamine) is 1-20 mM (preferably 8-12 mM, more preferably 9-10 mM); the concentration of the catalyst HAc (acetic acid) is 1-20 mM (preferably 5-10 mM, more preferably 6-8 mM); the solvent in the derivatization system is acetonitrile, or a mixture of acetonitrile and water, and the volume ratio of acetonitrile is 30%-100% (preferably 30%-60%, more preferably 50%-60%).

[0015] The amount of the derivatization system sprayed onto one side of the tissue section is 10–100 nmol / cm², preferably 20–50 nmol / cm².

[0016] The matrix is ​​α-cyano-4-hydroxycinnamic acid (CHCA) with a mass concentration of 6-10 mg / mL (preferably 7-8 mg / mL, more preferably 7 mg / mL); the solvent is an acetonitrile aqueous solution with a volume concentration of 40-70% (preferably 50%-60%, more preferably 60%), containing 0.1-0.5% (preferably 0.1-0.2%, more preferably 0.2%) trifluoroacetic acid; the spraying amount on one side of the tissue section is 0.07-0.5 mg / cm², more preferably 0.1-0.3 mg / cm², and most preferably 0.15-0.25 mg / cm².

[0017] The tissue sections are animal tissue sections, including mouse brain tissue sections or rat brain tissue sections;

[0018] Beneficial technical effects:

[0019] (1) The derivatization reagent used in the above technical solution not only contains a permanent positive charge but also enhances the ultraviolet absorption of the analyte at 335 nm, which can significantly change the molecular properties after derivatization and enhance the detection sensitivity of MALDI.

[0020] (2) The derivatization conditions are mild and the reaction steps are simple. The reaction is carried out under very mild conditions and occurs rapidly at ambient temperature and pressure without any stirring or agitation. It is suitable for in situ derivatization analysis of tissues and can avoid tissue dissolution and delocalization of target compounds.

[0021] (3) The above-mentioned technical methods can realize mass spectrometry imaging detection of various thiol molecules, realize in-situ MALDI mass spectrometry imaging analysis of compounds in tissues, and the four molecules belong to the GSH synthesis and metabolism pathway, which helps to solve the mechanism of GSH-related pathways and has good practical application value.

[0022] This invention can effectively improve the detection sensitivity of endogenous and exogenous thiol compounds and realize the visual analysis of thiol compounds in biological tissues, thus having good practical application value. Attached Figure Description

[0023] Figure 1 The standard curves (0.01-1 μg / mL) of GSH derivatized products labeled with the two derivatizing reagents in Example 1 are shown.

[0024] Figure 2 (a) and (b) are the mass spectrometry imaging results of GSH labeled with the two derivatization reagents in Example 2 on mouse brain tissue sections, respectively. Figure 2 (c) and (d) are mass spectra of the labeling results of the two derivatization reagents under MALDI-MS and MALDI-MSI conditions, respectively;

[0025] Figure 3 The mass spectrometry imaging results of four thiol compounds labeled with two derivatization reagents in Example 2 (Cys, Cys-Gly, γ-Glu-Cys and GSH);

[0026] Figure 4 The results show the trends of four thiol compounds in different mouse brain regions in the CSDS and CON groups in Example 3. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] Preparation of derivatizing reagents:

[0029]

[0030] 2,4,6-Trimethylpyran salt (0.5 mmol) and N-(2-aminoethyl)maleimide (0.5 mmol) were sequentially added to 10 mL of dichloromethane solution. After complete dissolution, triethylamine (0.5 mmol) was added. The mixture was stirred at room temperature for 10 min, followed by the addition of acetic acid (1 mmol), and the reaction was continued for 2 h. The reaction mixture was lyophilized and redissolved in 2-3 mL of dichloromethane (DCM). The insoluble matter was removed by centrifugation, and the crude product was extracted with diethyl ether. Finally, the crude product was purified by reversed-phase chromatography (RPC) (phase A: acetonitrile, phase B: water, linear elution at a gradient of 10%-40% in phase A over 0-30 min). The eluent was lyophilized to obtain the product. The product was a white solid. The structure of the compound was confirmed by nuclear magnetic resonance (H1N and C1N spectra), and the molecular weight was confirmed by mass spectrometry. The final structure of the compound was confirmed as [Ⅳ]PM1.

[0031] Following the same method described above, but differing in that equimolar amounts of 2,4,5-triphenylpyran salt and N-(2-aminoethyl)maleimide were used to synthesize compound [V]PM5. The product was a white solid, and the structure of the compound was confirmed as [V] by nuclear magnetic resonance (H1N, C1N) and mass spectrometry.

[0032] Composition of the derivatization system solution:

[0033] The derivatization system solution contains the above derivatization product at a final concentration of 0.1 mM, triethylamine at a final concentration of 10 mM, and acetic acid at a final concentration of 7 mM, wherein the solvent is acetonitrile:water = 1:1 (V / V).

[0034] Tissue section derivatization system solution spraying:

[0035] Tissue sections are mainly frozen tissues or organs. After freezing at -80℃ for 24 hours, they are cut into 14μm thick sections using a cryostat at -20℃ and mounted on ITO-coated glass slides. The sections are then dehydrated in a vacuum drying oven at 60℃ for 30 minutes. The area of ​​the sampled tissue sections is 2-80 mm². 2 The derivatizing solution is sprayed onto the tissue surface, with each three layers dried until all layers are applied. The final spray concentration of PM1 or PM5 on one side of the tissue section is maintained at 20–50 nmol / cm². 2 .

[0036] Matrix spraying;

[0037] An α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution (7 mg / mL α-cyano-4-hydroxycinnamic acid, containing 60% acetonitrile, 0.2% TFA, and the remainder water) was sprayed onto the tissue surface. Three layers were sprayed and dried before application until all layers were applied. The final spray concentration on one side of the tissue section was 0.15–0.25 mg / cm³. 2 .

[0038] Mass spectrometry signals of thiol compound derivatization products on tissue sections were acquired using a laser desorption / ionization source (MALDI).

[0039] The pre-processed slices were stored at -80℃ until MALDI data acquisition. During MALDI acquisition, the area to be acquired was selected for automatic sampling, with a sampling step size of 100μm, a laser spot size of 'medium', and an acquisition quality range of m / z 100-1000.

[0040] Example 1

[0041] Comparison of MALDI-TOF standard curves for GSH labeled with two labeling reagents, PM1 and PM5.

[0042] Dilute the glutathione solution with water to prepare a series of standard solutions ranging from 0.1 to 10 μg / mL (0.1, 0.2, 0.5, 1, 2, 5, 10 μg / mL). Add 40 μL of acetonitrile, 10 μL of PM1 or PM5 (1 mM acetonitrile solution), 10 μL of triethylamine (100 mM aqueous solution), 10 μL of acetic acid (70 mM aqueous solution), 20 μL of water, and 10 μL of one standard solution from the glutathione series to a 1 mL EP tube in sequence. After addition, mix and shake to obtain two series of standard solutions containing PM1 or PM5 at different concentrations. Then, take 1 μL of each mixture and apply it to a MALDI target plate. After the mixture dries, add 1 μL of the CHCA matrix described above. After drying, proceed with MALDI detection. Detection range: m / z 100-1000.

[0043] Figure 1 The products of GSH labeled with two derivatization reagents, PM1 and PM5, were presented, and standard curves of the derivatized products in the range of 0.01–1 μg / mL were further tested using MALDI-TOF. The fitted curve for the PM1 derivative was y = 94.99484x + 2253.099, R² = 0.99828; the fitted curve for the PM5 derivative was y = 82.72746x + 2415.315, R² = 0.99571. Both derivatization reagents showed good linearity in the GSH labeling range of 0.01–1 μg / mL, and the labeling sensitivity of PM5 was significantly better than that of PM1. Both labeling reagents can achieve the derivatization labeling of glutathione.

[0044] Example 2

[0045] Comparison of mass spectrometry imaging results after labeling thiol compounds with two derivatization reagents

[0046] Mice were euthanized by cervical dislocation after perfusion, and their brains were stored at -80°C for 24 hours. Then, 14 μL thick sections were cut using a cryostat and vacuum-dried. The area of ​​the derived tissue is statistically analyzed as follows: Figure 2 (a) The midhippocampus, thalamus, and cortex were 4 mm in diameter. 2 ; Figure 2 (b) The four hemispheres are 45, 50, 51 and 55 mm respectively. 2 ; Figure 3 (b) Each row is the same slice, with corresponding areas of 45, 50, 51 and 55 mm², respectively. 2 The derivatizing solution (composition: 0.1 mM PM1 or PM5, 10 mM triethylamine, 7 mM acetic acid, acetonitrile:water = 1:1 (V / V)) was evenly sprayed onto the surface of the tissue section using a sprayer. After every three layers, the solution was sprayed and dried, ultimately maintaining a coating density of 30 nmol / cm² on one side of the tissue section. 2 After completion, continue spraying a CHCA solution (CHCA concentration 7 mg / mL, containing 0.2% TFA, 60% acetonitrile, and the remainder water) onto the surface of the sections using a sprayer. Spray dry every three layers, ultimately achieving a spray coverage of 0.2 mg / cm² on one side of the tissue section. 2 After spraying, the sample is ready for MALDI analysis. The mass spectrometry imaging step size is 100 μm, the laser spot size is 'medium', and the detection range is m / z 100-1000.

[0047] Three different brain regions were observed on mouse brain slices: hippocampus (HP), cortex (CTX), and thalamus (TH). Figure 2 a) Point sampling and MALDI-TOF detection were used to obtain... Figure 2The mass spectrum shown in c (PM5 label) indicates that the labeled GSH (m / z 738.19) has high sensitivity and is the strongest signal in the mass spectrum. Other high-response peaks correspond to derivatization product fragments (m / z 334.16) and derivatization reagent peaks (m / z 431.17). Figure 2 c shows the mass spectrometry distribution of GSH in two brain regions after derivatization with two different derivatization reagents, with the PM5 derivatized product exhibiting a stronger mass spectrometry response. As shown in Table 1, we calculated the molecular weights of other corresponding thiol compounds and found their corresponding image distributions in the mass spectrometry imaging. Figure 3 The four thiol compounds we detected were all located on the GSH synthesis and metabolism pathway (Figure 3a). The PM1-labeled and PM5-labeled products had roughly the same spatial distribution, but mass spectrometry response signal analysis revealed that PM5 had a stronger sensitizing effect, corresponding to... Figure 3 Results b show that the signal intensity of PM5 is significantly better than that of PM1 in the same brain subregion.

[0048] Table 1. Changes in molecular weight of four thiol compounds before and after labeling with two derivatization reagents.

[0049]

[0050] Example 3

[0051] The changes in the content of four thiol compounds in different brain regions of the CSDS model were statistically analyzed. Brain samples from both the Chronic Social Frustration (CSDS) mouse model and the control mouse model (CON) were collected for comparison of changes in thiol compound content in the mouse brain. The pretreatment procedures for both mouse models were identical. The brain slice areas for CSDS and CON were 55 and 60 mm², respectively. Seven brain subregions were analyzed after sampling. Mice were euthanized by perfusion and cervical dislocation, and their brains were stored at -80℃ for 24 h. Then, 14 μL thick slices were cut using a cryostat and vacuum-dried. The area of ​​derivatized tissue is statistically analyzed as follows: The derivatizing solution (composition: 0.1 mM PM5, 10 mM triethylamine, 7 mM acetic acid, acetonitrile:water = 1:1 (V / V)) was uniformly sprayed onto the surface of the tissue sections using a sprayer. Each three layers were spray-dried, ultimately maintaining a spray coverage of 40 nmol / cm² on one side of the tissue section. Afterward, a CHCA solution (CHCA concentration 7 mg / mL, containing 0.2% TFA, 60% acetonitrile, and the remainder water) was sprayed onto the tissue section surface using a sprayer, again spray-dried every three layers, ultimately maintaining a spray coverage of 0.25 mg / cm² on one side of the tissue section. 2 After spraying, the sample is ready for MALDI analysis. The mass spectrometry imaging step size is 100 μm, the laser spot size is 'medium', and the detection range is m / z 100-1000.

[0052] Figure 4 We present the results of mouse brain mass spectrometry imaging obtained after treatment with PM5 derivatization reagent. We compared the changes in the ratios of four thiol compounds between the CSDS and CON groups. Finally, we analyzed the changes in seven relevant brain regions and found that the hippocampus was most affected, while other brain regions showed slight changes.

Claims

1. A mass spectrometry imaging method for detecting thiol compounds in tissues, characterized in that, The method includes: A derivatizing reagent with a special framework is used. The structure of the derivatizing reagent is as follows: ； R1-R5 are each independently hydrogen or one or more of the substituents methyl, ethyl, and phenyl, and the number of substituents is 0, one, two, three, four, or five; the length of the alkyl chain is 5≥n≥0, the phenyl contains one or more of C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, and halogen, the number of substituents is 1-5, and the halogen is one or more of F, Cl, Br, and I; (2) The derivatization reagent was prepared into a derivatization system solution and sprayed onto the surface of the tissue section to carry out the derivatization reaction. After the reaction was completed, the mercapto compound derivatization product was obtained. (3) Spray the above-derivatized tissue sections with matrix to obtain tissue section samples that can be used for mass spectrometry imaging; (4) Mass spectrometry signals of thiol compound derivatization products on tissue sections were collected using a laser desorption / ionization source (MALDI) to identify thiol compounds in the tissue.

2. The mass spectrometry imaging detection method for thiol compounds in tissues as described in claim 1, characterized in that, The synthesis process of the derivatization reagent is as follows: pyranium salt and N-substituted maleimide are mixed in a solvent and reacted at 15-40℃ for 5-60 min with an alkaline catalyst, and then reacted at 15-40℃ for 0.5-4 h with an acid catalyst to obtain the derivatization reagent. The pyranium salt wherein R1 to R5 are independently hydrogen or one or more of the substituents methyl, ethyl, and phenyl, and the number of substituents is 0, one, two, three, four, or five; the anion is BF4-; the phenyl group contains one or more of the substituents C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, and halogen, and the number of substituents is 1-5; the carbon chain length of the maleimide is 5≥n≥0; ； [Ⅱ] General structural formula of pyranium salts; [Ⅲ] General formula for N-substituted maleimide structure.

3. The mass spectrometry imaging detection method for thiol compounds in tissues as described in claim 2, characterized in that, The reaction solvent for the pyranium salt and N-substituted maleimide is an organic reagent, which is one or a combination of two or more of DCM, ACN, MeOH, THF, DMF and chloroform; The molar ratio of pyranium salt to N-substituted maleimide is 2:1 to 1:2, and the molar concentration of pyranium salt in the organic reagent is 0.01M to 0.3M.

4. The mass spectrometry imaging detection method for thiol compounds in tissues as described in claim 2 or 3, characterized in that, The acid-base catalyst is a base catalyst and an acid catalyst. In the specific reaction, the base is added first to react, and then the acid is added to continue the reaction. The base catalyst is one or two of triethylamine or N,N-diisopropylethylamine, and the acid catalyst is one, two or three of formic acid, acetic acid or benzoic acid. The molar ratio of the acid-base catalyst to the pyranium salt is 1:10-10:

1.

5. The mass spectrometry imaging detection method for thiol compounds in tissues as described in claim 1, characterized in that, The derivatization system is prepared by mixing derivatization reagent, acetic acid and triethylamine, spraying directly onto the surface of tissue sections, and allowing it to air dry without incubation. In the derivatization system, the concentration of the derivatizing reagent is 0.05-5 mM; the concentration of the catalyst TEA (triethylamine) is 1-20 mM; the concentration of the catalyst HAc (acetic acid) is 1-20 mM; and the solvent in the derivatization system is acetonitrile, or a mixture of acetonitrile and water, with the volume ratio of acetonitrile being 30%-100%.

6. The mass spectrometry imaging detection method for thiol compounds in tissues as described in claim 1, characterized in that, The area of ​​the sampled tissue section is 2-80 mm. 2 .

7. The mass spectrometry imaging method for detecting thiol compounds in tissues as described in claim 1 or 6, characterized in that, The amount of derivatizing reagent sprayed onto one side of the tissue section is 10–100 nmol / cm². 2 .

8. The mass spectrometry imaging method for detecting thiol compounds in tissues as described in claim 1 or 6, characterized in that, The matrix is ​​α-cyano-4-hydroxycinnamic acid (CHCA) with a mass concentration of 6–10 mg / mL; the solvent is an acetonitrile aqueous solution with a volume concentration of 40–70%, containing 0.1–0.5% trifluoroacetic acid; the spraying amount on one side of the tissue section is 0.07–0.5 mg / cm². 2 .

9. The mass spectrometry imaging detection method for thiol compounds in tissues as described in claim 1, characterized in that, The tissue sections are animal tissue sections, including mouse brain tissue sections or rat brain tissue sections.

10. The mass spectrometry imaging detection method for thiol compounds in tissues as described in claim 1 or 9, wherein the thiol compound is one or more polypeptides containing thiol groups in the tissue; Mass spectrometry signals of thiol compound derivatization products on tissue sections were acquired using a laser desorption / sorption ionization source (MALDI). The thiol compounds in the tissue were then identified, and the mass-to-charge ratio information of the derivatization products was extracted to obtain mass spectrometry images.

Images