Method for in situ derivatization of cholesterol in tissue sections and MALDI mass spectrometry imaging and applications thereof

Abstract

The application provides a mass spectrometry imaging detection method for cholesterol in a tissue slice and application thereof, 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 a cholesterol extraction solvent and a derivatization system solution to the surface of a prepared tissue slice to obtain a derivatization product of cholesterol; and performing matrix spraying on the tissue slice after derivatization and performing MALDI mass spectrometry imaging analysis. The application can effectively improve the detection sensitivity of cholesterol in a tissue, realizes visual analysis of cholesterol in a biological tissue, and therefore has good practical popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of mass spectrometry detection technology, and relates to a MALDI mass spectrometry imaging method and its application that can detect cholesterol in biological tissues with high sensitivity in positive ion mode. Background Technology

[0002] Cholesterol is a crucial component of cell membranes, playing a vital role in maintaining membrane structure and transmembrane signal transduction. The brain has the highest cholesterol content among mammals, with the human brain containing up to 25% total cholesterol and cholesterol derivatives; in mice, the brain contains approximately 15% total cholesterol. There are two main types of cholesterol in the central nervous system (CNS): one composed of the myelin sheath of oligodendrocytes, accounting for 70% of total CNS cholesterol; and the other composed of the plasma membranes of astrocytes and neurons. Furthermore, cholesterol is a precursor compound for hydroxysteroids, steroid hormones, and bile acids. Normally, the brain has coordinated regulatory mechanisms to maintain stable cholesterol levels and estrogen levels for normal function. However, defects in cholesterol metabolism can lead to structural and functional impairments of the CNS, such as Niemann-Pick disease, Huntington's disease, Alzheimer's disease, and Parkinson's disease. These diseases are associated to varying degrees with cholesterol biosynthesis and lipid transport. Therefore, in-depth research into cholesterol and its metabolism may provide new insights into the complex molecular processes underlying neurodegenerative diseases.

[0003] Mass spectrometry, as an important analytical technique, is increasingly widely used in the detection of low-concentration compounds. For cholesterol, several methods have been developed for qualitative and quantitative detection using derivatization combined with MS technology. While LC-MS / MALDI-MS combined with derivatization can achieve highly sensitive cholesterol detection, the results we obtained are average values ​​that do not account for the loss of spatial relationships within tissues due to tissue heterogeneity. By combining the differences in cholesterol-sterol abundance with spatial distribution, we can better understand the biochemical and physiological roles of sterols. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) is a novel multi-molecule imaging technique based on mass spectrometry (MS) that can simultaneously image multiple biomolecules in cells and tissues without labeling. Compared with traditional mass spectrometry techniques, mass spectrometry imaging can provide more spatial information, not just changes in the average content of biological samples, and is more meaningful for distinguishing local differences in tissues and organs.

[0004] Over the past decade, various auxiliary desorption methods have been developed for the analysis of cholesterol and endogenous lipids, including nanostructured initiator MS, sputtered silver MALDI, CBS-Au-LDI, and silver nanoparticle MALDI. However, compared with other high-abundance lipids, cholesterol, with its low proton affinity and low acidity, exhibits insufficient sensitivity in MALDI-MS detection. Histochemical derivatization (OTCD) methods can effectively enhance the detection of cholesterol by MALDI-MS. Manicke et al. directly incorporated the charge-labeled reagent betaine aldehyde into DESI solvent spray and then visualized cholesterol through in-situ chemical derivatization. Angelini et al. used an enzyme-assisted derivatization strategy in tissues to improve the detection of cholesterol in brain tissue sections, first using cholesterol oxidase to oxidize the C3-hydroxyl group of cholesterol to a carbonyl group, followed by derivatization with Girard's reagent. However, complex pretreatment and lengthy incubation may introduce more uncertainties into mass spectrometry imaging. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a mass spectrometry imaging method for detecting cholesterol in tissues. The present invention synthesizes a derivatization reagent that can be applied to MALDI mass spectrometry imaging of compounds containing o-dihydroxyl groups in situ, significantly improving the detection sensitivity of compounds containing o-dihydroxyl groups in tissues, and realizing the visual analysis of endogenous metabolites and exogenous drugs containing o-dihydroxyl functional groups in biological tissues. The derivatization reagent is synthesized by reacting the oxonium ions of pyranium salts with the primary amine of aminomethylphenylboronic acid salt to synthesize the N-alkylpyridinium derivative pyridinium-methylphenylboronic acid. The synthesis is carried out in a methanol-water solution, and the reaction is completed immediately without heating or prolonged stirring; only a small amount of organic base is added as an oxidant. Furthermore, there is no need to separate and purify the synthesized pyridinium-methylphenylboronic acid from the synthesis solution system; the synthesis solution system can be directly sprayed onto the surface of tissue sections for mass spectrometry imaging detection of cholesterol.

[0006] In this invention, we developed a novel class of pyranonium salt-based derivatization reagents for cholesterol visualization. Building upon previous work, we reacted highly reactive pyranonium salts with neutral amino acids to activate the carboxyl group via a permanent positive charge, followed by rapid cholesterol labeling under mild conditions in the presence of cross-linking agents EDCl and HOBt. Our proposed method has been successfully applied to MSI of cholesterol in the mouse brain. This method has also been successfully applied to mass spectrometry imaging of cholesterol in the brain of an SOD1 mutant mouse model, elucidating the trends in cholesterol changes in different brain regions at different stages of the disease.

[0007] The first part of this invention provides a mass spectrometry imaging method for detecting cholesterol in tissues, the method comprising at least:

[0008] (a) In solution, N-alkylpyridinium derivative carboxymethylpyridinium is generated by the reaction of oxonium ions of pyranium salt with the primary amine of neutral amino acid. The derivative is separated and purified by high performance liquid chromatography, and the fraction is lyophilized for later use.

[0009] (b) The extraction reagent was sprayed onto the surface of the tissue section attached to the ITO slide, and the section was allowed to dry naturally to extract cholesterol.

[0010] (c) Dissolve the derivatization reagent carboxymethylpyridinium in an organic solvent and mix it with a catalyst to prepare a derivatization system solution. Spray the derivatization system onto the surface of a tissue section and allow it to dry naturally to perform cholesterol derivatization. That is, the cholesterol C3 hydroxyl group and the carboxyl group of carboxymethylpyridinium undergo an esterification reaction under the action of a catalyst, transferring the permanent positive charge of carboxymethylpyridinium to cholesterol to obtain the cholesterol derivatization product.

[0011] (d) The tissue sections after the above derivatization treatment are coated with matrix to obtain tissue section samples that can be used for mass spectrometry imaging;

[0012] (e) Mass spectrometry signals of derivatized products were acquired using a laser desorption / ionization source (MALDI) to qualitatively identify cholesterol in tissues and extract mass-to-charge ratio information of derivatized products to obtain mass spectrometry images.

[0013] As a further embodiment of the present invention: the derivatization reagent is synthesized by mixing a pyranium salt solution and an amino acid solution, adding an organic base catalyst, mixing thoroughly, stirring at room temperature for 10 minutes, adding acetic acid, and stirring at room temperature for 2 hours to obtain a carboxymethylpyridinium solution as the derivatization reagent.

[0014] As a further embodiment of the present invention: R1 to R5 of the pyranium salt can be one, two or three of hydrogen and substituents methyl, phenyl, benzyl, and the number of substituents can be one, two, three, four or five, and the anion is BF4.

[0015] As a further embodiment of the present invention: the amino acid may be one of glycine, alanine and γ-aminobutyric acid;

[0016] As a further embodiment of the present invention: the concentration of the pyranium salt solution is 0.5-5 mM, preferably 1-3 mM, more preferably 1 mM, and the concentration of the amino acid solution is 0.5-5 mM, preferably 2-3 mM, more preferably 2 mM; in the mixed solution synthesis system, the molar ratio of pyranium salt to amino acid is 10:1-1:1, preferably 8:1-2:1, more preferably 2:1;

[0017] As a further embodiment of the present invention: the solvent for the pyranium salt and the amino acid is an aqueous solution of acetonitrile, preferably, the volume concentration of acetonitrile in the aqueous solution is 25-80%, more preferably 50-75%, and even more preferably 60%;

[0018] As a further embodiment of the present invention: the organic base catalyst is one or two of triethylamine or N,N-diisopropylethylamine; the final concentration of the organic base in the synthesis system is 0.1-1 mM, preferably 0.5-0.75 mM, more preferably 0.5 mM;

[0019] As a further embodiment of the present invention: the final concentration of acetic acid in the synthesis system is 0.2-2 mM, preferably 0.5-1.5 mM, and more preferably 1 mM;

[0020] As a further aspect of the present invention: the extraction solvent for cholesterol on the surface of tissue sections can be one of an aqueous solution of acetonitrile, methanol, or ethanol, more preferably 80%, and the spraying amount on the surface of the tissue sections is 5–30 µL / cm. 2 More preferably 10 µL / cm 2 ;

[0021] As a further embodiment of the present invention: the derivatization system solution is prepared by mixing the derivatization reagent and catalyst 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), 1-hydroxybenzotriazole (HOBt) and triethylamine, and spraying it directly onto the surface of the tissue section without incubation and allowing it to dry naturally.

[0022] As a further aspect of the present invention: in the derivatization system solution, the final concentration of the derivatization reagent is 0.5-5 mM, preferably 1-3 mM, and more preferably 2 mM;

[0023] As a further aspect of the present invention: in the derivatization system, the final concentration of the catalyst EDCl is 0.5-5 mM, preferably 1-3 mM, and more preferably 1.2 mM;

[0024] As a further aspect of the present invention: in the derivatization system, the final concentration of the catalyst HOBt is 0.5-5 mM, preferably 1-3 mM, and more preferably 1.2 mM;

[0025] As a further aspect of the present invention: in the derivatization system, the final concentration of the catalyst TEAl is 1-8 mM, preferably 3-5 mM, and more preferably 3.6 mM;

[0026] As a further aspect of the present invention: in the derivatization system solution, the derivatization reagent, EDCl and HOBt solvent are acetonitrile;

[0027] As a further embodiment of the present invention: the amount of derivatization reagent sprayed onto one side of the tissue section is 10–100 nmol / cm². 2 Preferably 20–50 nmol / cm 2 More preferably 25 nmol / cm 2 ;

[0028] As a further embodiment of the present invention: the matrix is ​​α-cyano-4-hydroxycinnamic acid (CHCA) with a mass concentration of 5-10 mg / mL, preferably 7 mg / mL; the solvent is an acetonitrile aqueous solution with a volume concentration of 40-60%, preferably 60%; it contains trifluoroacetic acid at a concentration of 0.1-0.2%, preferably 0.2%; and the spraying amount on one side of the tissue section is 0.07-0.35 mg / cm². 2 Preferably, the concentration is 0.15–0.25 mg / cm³. 2 Alternatively, the matrix may be trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malonium (DCTB) with a mass concentration of 7–35 mg / mL, preferably 10–20 mg / mL, more preferably 10 mg / mL, and the solvent may be dichloromethane or a mixture of dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 100–50:0–50, and the spraying amount on one side of the tissue section may be 0.1–0.5 mg / cm². 2 Preferably, the concentration is 0.15–0.25 mg / cm³. 2 ;

[0029] As a further aspect of the present invention: the tissue sections are selected as animal tissue sections, including mouse brain tissue sections or rat brain tissue sections;

[0030] (1) The derivatization reagents used in the above technical solution are synthesized in a solution system. The operation is simple and does not require heating or incubation. The synthesized derivatization reagents do not need to be separated and prepared. The derivatization reagent mixture can be used for derivatization.

[0031] (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 cholesterol derivatization analysis in tissues, avoiding tissue dissolution and delocalization of the target compound.

[0032] (3) The above technical methods introduce permanent positive charges into the structure of cholesterol by derivatizing cholesterol, thereby improving the sensitivity of cholesterol MALDI mass spectrometry detection and realizing in situ MALDI mass spectrometry imaging analysis of cholesterol in tissues, which has good practical application value. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0034] Figure 1 This is the mass spectrometry characterization of 1-(carboxymethyl)-2,4,6-trimethylpyridinium in Example 1 of the present invention.

[0035] Figure 2 This is the synthesis reaction formula of the derivatization reagent in Example 1 of the present invention.

[0036] Figure 3 This is the LC-MS / MS mass spectrum of the cholesterol derivatization product in solution system in Example 2 of the present invention.

[0037] Figure 4 This is a mass spectrometry image of endogenous cholesterol in mouse brain tissue slices under different extraction conditions in Example 3 of the present invention.

[0038] Figure 5 This is a cholesterol mass spectrometry image of a coronal section of mouse brain tissue in Example 4 of the present invention.

[0039] Figure 6 This is the MALDI-derived intra-source cleavage reaction formula of the cholesterol derivatization product in Example 5 of the present invention.

[0040] Figure 7 This is a mass spectrometry image of cholesterol on the surface of a sagittal section of mouse brain tissue in Example 5 of this invention.

[0041] Figure 8 This is a mass spectrometry image of cholesterol on the surface of a coronal section of the brain tissue of a SOD1-G93A mouse in Example 6 of this invention. Detailed Implementation

[0042] Example 1: Synthesis of 1-(carboxymethyl)-2,4,6-trimethylpyridinium

[0043] (1) Accurately weigh 21 mg of 2,4,6-trimethylpyranium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 2,4,6-trimethylpyranium tetrafluoroborate (20 mM) solution for later use;

[0044] (2) Accurately weigh 7.5 mg of glycine, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain glycine (20 mM) solution for later use;

[0045] (3) 1 mL each of 2,4,6-trimethylpyranium tetrafluoroborate solution (20 mM) and glycine solution (20 mM) were added sequentially to 18 mL of 60% acetonitrile (ACN / H2O = 6:4, volume ratio), vortexed, and triethylamine (final concentration 1 mM) was added. After stirring at room temperature for 10 minutes, acetic acid (final concentration 2 mM) was added, and the reaction was continued for 2 hours. The reaction mixture was lyophilized and redissolved in dichloromethane, the insoluble matter was removed by centrifugation, and the crude product was obtained by extraction with diethyl ether. The crude product was separated by high-performance preparative liquid chromatography (HPLC) using an Agilent Poroshell 120 EC-C18 column (100 mm × 3.0 mm, 2.7 μm). Mobile phase A was water (containing 0.5% formic acid, v / v), and mobile phase B was acetonitrile (containing 0.5% formic acid, v / v). Elution gradient was 0–8 min, 2% B–100% B. The peak at retention time 1.75–2 min was collected. The eluent was freeze-dried to obtain 1-(carboxymethyl)-2,4,6-trimethylpyridinium. The product was characterized by LC-ESI-QTOF-MS. Figure 1 The m / z of 1-(carboxymethyl)-2,4,6-trimethylpyridinium is 180.0883, which is consistent with the theoretical value.

[0046] Example 2: Derivatization of cholesterol in solution and LC-MS / MS mass spectrometry analysis

[0047] (1) Accurately weigh 21 mg of 2,4,6-trimethylpyranium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain a 2,4,6-trimethylpyranium tetrafluoroborate (20 mM) solution for later use;

[0048] (2) Accurately weigh 13.6 mg of 2,6-dimethyl-4-phenylpyridinium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain a 10 mM solution of 2,6-dimethyl-4-phenylpyridinium tetrafluoroborate.

[0049] (3) Accurately weigh 19.8 mg of 2,4,5-triphenylpyridinium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 2,4,5-triphenylpyridinium tetrafluoroborate (10 mM) solution for later use;

[0050] (4) Accurately weigh 21.2 mg of 2-phenyl-4,5-diphenylmethylpyridinium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 2-phenyl-4,5-diphenylmethylpyridinium tetrafluoroborate (10 mM) solution for later use;

[0051] (5) Accurately weigh 23.6 mg of 2,3,4,5-tetraphenylpyridinium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain a 10 mM solution of 2,3,4,5-tetraphenylpyridinium tetrafluoroborate.

[0052] (6) Accurately weigh 3.75 mg of glycine, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain glycine (10 mM) solution for later use;

[0053] (7) Mix 1 mL of each pyranium tetrafluoroborate solution from steps (1) to (5) with 1 mL of glycine solution, and add them to 18 mL of 60% acetonitrile (ACN / H2O = 6:4, volume ratio). Vortex mix, add triethylamine (final concentration 1 mM), stir at room temperature for 10 min, add acetic acid (final concentration 2 mM), and continue the reaction for 2 hours. Freeze-dry the reaction mixture and redissolve it in dichloromethane. Centrifuge to remove insoluble matter, and extract with diethyl ether to obtain the crude product. The crude product was separated by high-performance preparative liquid chromatography (HPLC). The chromatographic column was an Agilent Poroshell 120 EC-C18 (100 mm × 3.0 mm, 2.7 μm). Mobile phase A was water (containing 0.5% formic acid, v / v), and mobile phase B was acetonitrile (containing 0.5% formic acid, v / v). Elution gradient was 0–8 min, 2% B–100% B. The carboxymethylpyridinium peak was collected at 1.75–3 min. The eluents were freeze-dried to obtain 1-(carboxymethyl)-2,4,6-trimethylpyridinium, 1-(carboxymethyl)-2,6-dimethyl-4-phenylpyridinium, 1-(carboxymethyl)-2,4,5-triphenylpyridinium, 1-(carboxymethyl)-2-phenyl-4,5-diphenylmethylpyridinium, and 1-(carboxymethyl)-2,3,4,5-tetraphenylpyridinium products.

[0054] (8) Dissolve the series of derivatizing reagents prepared in step (7) in acetonitrile to a final concentration of 2 mM, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (final concentration 1.2 mM), 1-hydroxybenzotriazole (final concentration 1.2 mM) and triethylamine (final concentration 3.6 mM) in sequence to obtain the derivatization system solution.

[0055] (9) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (final concentration 1.2 mM), 1-hydroxybenzotriazole (final concentration 1.2 mM) and triethylamine (final concentration 3.6 mM) to acetonitrile in sequence to obtain a control system solution without derivatization reagent.

[0056] (10) Accurately weigh 1 mg of cholesterol, add methanol aqueous solution (80:20, volume ratio), vortex to mix, sonicate for 5 minutes to obtain cholesterol standard solution for later use;

[0057] (11) Mix 10 µL of the series of derivatization system solutions prepared in steps (8) and (9) above and 10 µL of the control system solution without derivatization reagent with 10 µL of cholesterol stock solution in step (10) to obtain a series of cholesterol derivatization product solutions.

[0058] (12) Using an Agilent 6520 Q-TOF mass spectrometer in positive ion mode, with a mass-to-charge ratio detection range of 140 to 1000, a series of cholesterol derivatization product solutions were detected.

[0059] (13) Based on the cholesterol derivatization reaction formula ( Figure 2 The mass spectrometry signals of the derivatized products were extracted using Bruker Agilent Masshunter software, with m / z values ​​of 548.5, 610.5, 734.5, 752.5, and 900.5, respectively. The results are as follows: Figure 3 As shown, when 1-(carboxymethyl)-2,4,6-trimethylpyridinium, 1-(carboxymethyl)-2,6-dimethyl-4-phenylpyridinium, 1-(carboxymethyl)-2,4,5-triphenylpyridinium, 1-(carboxymethyl)-2-phenyl-4,5-diphenylmethylpyridinium, and 1-(carboxymethyl)-2,3,4,5-tetraphenylpyridinium are used as derivatizing reagents, mass spectrometry signals of cholesterol derivatized products can be obtained. The relative abundance of cholesterol derivatized with 1-(carboxymethyl)-2,4,6-trimethylpyridinium is the highest. When the solution without derivatizing reagent is mixed with the cholesterol solution, no mass spectrometry signal of the cholesterol molecular ion peak and its fragment peaks can be detected.

[0060] Example 3: Derivatization and MALDI mass spectrometry analysis of endogenous cholesterol in mouse brain tissue sections

[0061] (1) Take the complete brain tissue of mice frozen at -80℃, and prepare four coronal sections of brain tissue with a thickness of 14µm using a slicer. Transfer the prepared brain tissue sections onto four indium tin oxide (ITO) glass slides and dry them in a vacuum desiccator at room temperature for 15 minutes.

[0062] (2) Accurately weigh 21 mg of 2,4,6-trimethylpyranium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 2,4,6-trimethylpyranium tetrafluoroborate (20 mM) solution for later use;

[0063] (3) Accurately weigh 3.75 mg of glycine, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain glycine (10 mM) solution for later use;

[0064] (4) Mix 1 mL of the 2,4,6-trimethylpyranium tetrafluoroborate solution prepared in step (2) with 1 mL of the glycine solution prepared in step (3), and add them separately to 18 mL of 60% acetonitrile (ACN / H2O=6:4, volume ratio), vortex to mix, and add triethylamine (final concentration 1 mM); stir at room temperature for 10 min, then add acetic acid (final concentration 2 mM), and continue the reaction for 2 hours. Freeze-dry the reaction mixture and redissolve it in dichloromethane, centrifuge to remove insoluble matter, and extract with diethyl ether to obtain the crude product. The crude product was separated by high performance preparative liquid chromatography (HPLC). The chromatographic column was an Agilent Poroshell 120 EC-C18 (100 mm × 3.0 mm, 2.7 μm). The mobile phase was water (containing 0.5% formic acid by volume) and acetonitrile (containing 0.5% formic acid by volume) by volume. The elution gradient was 0–8 min, 2% B–100% B. The chromatographic peak at the retention time of 1.75–2 min was collected. The eluent was freeze-dried to obtain 1-(carboxymethyl)-2,4,6-trimethylpyridinium.

[0065] (5) Dissolve the derivatization reagent prepared in step (4) in acetonitrile to a final concentration of 4 mM, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 mM), 1-hydroxybenzotriazole (1.2 mM) and triethylamine (3.6 mM) in sequence to obtain the derivatization system solution.

[0066] (6) Accurately weigh 35 mg of α-cyano-4-hydroxycinnamic acid (CHCA), add 5 mL of acetonitrile:water (60:40, v / v) solution containing 0.2% trifluoroacetic acid by volume, vortex mix, sonicate for 5 minutes to obtain CHCA (7 mg / mL) solution for later use;

[0067] (7) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 Place a brain tissue slice prepared in step (1) in the center of the spray chamber, with one side of the ITO slide tissue slice facing upwards. Spray 80% acetonitrile (v / v) into the spray chamber in 18 coats. Allow each coat to air dry at room temperature after spraying. A total of 1 mL of 80% acetonitrile (v / v) was used, resulting in a final spray volume of 10 µL / cm. 2 After the final spray coating, place the ITO glass slide in a vacuum desiccator and dry at room temperature for 15 minutes.

[0068] (8) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 Place another brain tissue slice prepared in step (1) in the center of the spray chamber, with one side of the ITO slide tissue slice facing upwards. Spray 80% ethanol (v / v) into the spray chamber in 18 coats, allowing each coat to air dry at room temperature. A total of 1 mL of 80% ethanol (v / v) was used, resulting in a final spray volume of 10 µL / cm. 2 After the final spray coating, place the ITO glass slide in a vacuum desiccator and dry at room temperature for 15 minutes.

[0069] (9) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 Place the third brain tissue slice prepared in step (1) in the center of the spray chamber, with one side of the ITO slide tissue slice facing upwards. Spray 80% methanol (v / v) into the spray chamber in 18 coats. Allow each coat to air dry at room temperature after spraying. A total of 1 mL of 80% methanol (v / v) was used, and the final spray volume was 10 µL / cm. 2 After the final spray coating, place the ITO glass slide in a vacuum desiccator and dry at room temperature for 15 minutes.

[0070] (10) The fourth brain tissue slice prepared in (1) was not sprayed with any cholesterol extraction reagent. The ITO slide was placed directly in a vacuum desiccator and dried at room temperature for 15 minutes.

[0071] (11) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 The brain tissue sections prepared in steps (7) to (10) above, with one side of the ITO slide tissue section facing upwards, were placed in the center of the spraying chamber. The derivatization system solution obtained in step (5) was sprayed onto the tissue in 18 sprays, using 1 mL of solution each time, with a final spraying amount of 40 nmol / cm³. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide in a vacuum desiccator and dry it at room temperature for 15 minutes.

[0072] (12) Then use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 The brain tissue slices prepared in step (11) and the ITO slide tissue slices were placed in the center of the spray chamber with one side facing up. The CHCA solution prepared in step (6) was sprayed into the spray chamber in 24 sprays, using 1.5 mL of solution each time. The final CHCA spraying amount was 0.1 mg / cm³. 2After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide with the side containing the drying point of the standard sample facing up in a vacuum desiccator and dry at room temperature for 15 minutes.

[0073] (13) Using a Bruker UltraFlex Ⅲ MALDI-TOF / TOF mass spectrometer, in positive ion and reflection mode, with a mass-to-charge ratio detection range of 140 to 1000, mass spectrometry analysis was performed on one surface of an ITO slide containing the drying point of the standard.

[0074] (14) Based on the cholesterol derivatization reaction formula ( Figure 2 The mass spectrometry signal of the derivatized product was extracted using Bruker DataAnalysis software, with an m / z value of 548.5. The results are as follows: Figure 4 As shown, mass spectrometry signals of cholesterol derivatization products on mouse brain tissue sections can be obtained when 80% acetonitrile, 80% methanol, and 80% ethanol are used as cholesterol extraction reagents from the surface of mouse brain tissue, 1-(carboxymethyl)-2,4,6-trimethylpyridinium is used as the derivatization reagent, and CHCA is used as the matrix. 80% ethanol has the strongest ability to extract cholesterol from the surface of tissue sections. No cholesterol derivatives or cholesterol mass spectrometry signals are found in mouse brain tissue sections that have not been extracted with organic solvents.

[0075] Example 4: Cholesterol Derivatization and MALDI Mass Spectrometry Imaging Analysis on Coronal Sections of Mouse Brain Tissue

[0076] (1) Take the complete brain of a mouse frozen at -80℃, and prepare two coronal sections of brain tissue with a thickness of 14µm using a slicer. Transfer the prepared brain tissue sections onto two indium tin oxide (ITO) glass slides and dry them in a vacuum desiccator at room temperature for 15 minutes.

[0077] (2) Accurately weigh 42 mg of 2,4,6-trimethylpyranium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 2,4,6-trimethylpyranium tetrafluoroborate (40 mM) solution for later use;

[0078] (3) Accurately weigh 3.75 mg of glycine, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain glycine (10 mM) solution for later use;

[0079] (4) Mix 1 mL of the 2,4,6-trimethylpyranium tetrafluoroborate solution prepared in step (2) with 1 mL of the glycine solution prepared in step (3), and add them to 18 mL of 60% acetonitrile (ACN / H2O=6:4). Vortex to mix, and add triethylamine (1 mmol). Stir at room temperature for 10 min, then add acetic acid (2 mmol) and continue the reaction for 2 h. Freeze-dry the reaction mixture and redissolve it in dichloromethane. Centrifuge to remove insoluble matter and extract with diethyl ether to obtain the crude product. The crude product was separated by high performance preparative liquid chromatography (HPLC). The chromatographic column was an Agilent Poroshell 120 EC-C18 (100 mm × 3.0 mm, 2.7 μm). The mobile phase was water (containing 0.5% formic acid by volume) and acetonitrile (containing 0.5% formic acid by volume) by volume. The elution gradient was 0–8 min, 2% B–100% B. The chromatographic peak at the retention time of 1.75–2 min was collected. The eluent was freeze-dried to obtain 1-(carboxymethyl)-2,4,6-trimethylpyridinium product.

[0080] (5) Dissolve the derivatizing reagent prepared in (4) in acetonitrile to a final concentration of 2.5 mM, and then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 mM), 1-hydroxybenzotriazole (1.2 mM) and triethylamine (3.6 mM) in sequence to obtain the derivatization system solution.

[0081] (6) Accurately weigh 50 mg of trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malonitrile (DCTB), add 5 mL of dichloromethane:methanol (50:50, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain DCTB (10 mg / mL) solution for later use.

[0082] (7) Accurately weigh 35 mg of α-cyano-4-hydroxycinnamic acid (CHCA), add 5 mL of acetonitrile:water (60:40, v / v) solution containing 0.2% trifluoroacetic acid by volume, vortex mix, sonicate for 5 minutes to obtain CHCA (7 mg / mL) solution for later use;

[0083] (8) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 Two mouse brain tissue slices prepared in step (1) were placed on ITO glass slides with one side of the tissue slice facing upwards in the center of the spray chamber. 80% methanol was sprayed into the spray chamber in 18 sprays, using 1 mL of solution each time, with a final spray volume of 10 µL / cm. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide in a vacuum desiccator and dry it at room temperature for 15 minutes.

[0084] (9) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 Two mouse brain tissue slices prepared in step (8) were placed in the center of the spray chamber with one side of the ITO slide tissue slice facing upwards. The derivatization system solution obtained in step (5) was sprayed into the spray chamber in 18 sprays, using 1 mL of solution each time, with a final spraying volume of 25 nmol / cm³. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide in a vacuum desiccator and dry it at room temperature for 15 minutes.

[0085] (10) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 A mouse brain tissue slice prepared in step (9) was placed in the center of the spray chamber with the side of the ITO slide containing the drying point of the standard facing upwards. The matrix solution prepared in step (6) was sprayed into the spray chamber in 24 sprays, using 3 mL of solution each time. The final CHCA spraying amount was 0.21 mg / cm³. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide with the side containing the drying point of the standard sample facing up in a vacuum desiccator and dry at room temperature for 15 minutes.

[0086] (11) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 Another mouse brain tissue slice prepared in (9) was placed in the center of the spray chamber with the side of the ITO slide containing the drying point of the standard facing upwards. The matrix solution prepared in step (7) was sprayed into the spray chamber in 24 sprays, using 3 mL of solution each time. The final spray amount of DCTB was 0.3 mg / cm³. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide with the side containing the drying point of the standard sample facing up in a vacuum desiccator and dry at room temperature for 15 minutes.

[0087] (12) Using a Bruker UltraFlex Ⅲ MALDI-TOF / TOF mass spectrometer, in positive ion and reflection mode, with a mass-to-charge ratio detection range of 140 to 1000, mass spectrometry analysis was performed on one side of the tissue section on an ITO slide.

[0088] (13) Based on the cholesterol derivatization reaction formula ( Figure 2 The mass spectrometry signal of the derivatized product was extracted using Bruker DataAnalysis software, with an m / z value of 548.5. The results are as follows: Figure 5As shown, when DCTB and CHCA are used as matrices and 1-(carboxymethyl)-2,4,6-trimethylpyridinium is used as the derivatization reagent, mass spectrometry signals of cholesterol derivatization products on mouse brain tissue slices can be obtained. The relative abundance of cholesterol derivatization products is higher when DCTB is used as the matrix, and cholesterol is more abundant in the white matter of mouse brain tissue than in the gray matter.

[0089] Example 5: Derivatization and MALDI mass spectrometry analysis of cholesterol in sagittal sections of mouse brain tissue

[0090] (1) Take a mouse brain frozen at -80℃ and prepare a sagittal section of brain tissue with a thickness of 14µm using a slicer. Transfer the prepared brain tissue section onto an indium tin oxide (ITO) glass slide and dry it in a vacuum desiccator at room temperature for 15 minutes.

[0091] (2) Accurately weigh 21 mg of 2,4,6-trimethylpyranium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 2,4,6-trimethylpyranium tetrafluoroborate (20 mM) solution for later use;

[0092] (3) Accurately weigh 7.5 mg of glycine, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain glycine (20 mM) solution for later use;

[0093] (4) Mix 1 mL of the 2,4,6-trimethylpyranium tetrafluoroborate solution prepared in step (2) with 1 mL of the glycine solution prepared in step (3), and add them to 18 mL of 60% acetonitrile (ACN / H2O=6:4). Vortex to mix, and add triethylamine (1 mmol). Stir at room temperature for 10 min, then add acetic acid (2 mmol) and continue the reaction for 2 h. Freeze-dry the reaction mixture and redissolve it in DCM. Centrifuge to remove insoluble matter and extract with diethyl ether to obtain the crude product. The crude product was separated by high performance preparative liquid chromatography (HPLC). The chromatographic column was an Agilent Poroshell 120 EC-C18 (100 mm × 3.0 mm, 2.7 μm). The mobile phase was water (containing 0.5% formic acid by volume) and acetonitrile (containing 0.5% formic acid by volume) by volume. The elution gradient was 0–8 min, 2% B–100% B. The chromatographic peak at the retention time of 1.75–2 min was collected. The eluent was freeze-dried to obtain 1-(carboxymethyl)-2,4,6-trimethylpyridinium product.

[0094] (5) Dissolve the derivatizing reagent prepared in (4) in acetonitrile to a final concentration of 2 mM, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 mM), 1-hydroxybenzotriazole (1.2 mM) and triethylamine (3.6 mM) in sequence to obtain the derivatization system solution.

[0095] (6) Accurately weigh 50 mg of trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malonitrile (DCTB), add 5 mL of dichloromethane:methanol (50:50, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain DCTB (10 mg / mL) solution for later use.

[0096] (7) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 The brain tissue sections prepared in step (1) are placed on ITO glass slides with one side facing up in the center of the spray chamber. 80% ethanol is sprayed into the spray chamber in 18 sprays, using 1 mL of solution each time, with a final spray volume of 10 µL / cm. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide in a vacuum desiccator and dry it at room temperature for 15 minutes.

[0097] (8) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 The brain tissue slices prepared in step (7) are placed with one side of the ITO slide facing upwards in the center of the spray chamber. The derivatization reagent solution obtained in step (5) is sprayed into the spray chamber in 18 sprays, using 1 mL of solution each time. The final spray volume is 20 nmol / cm³. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide in a vacuum desiccator and dry it at room temperature for 15 minutes.

[0098] (9) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 The brain tissue slices prepared in step (8) were placed in the center of the spray chamber with the side of the ITO slide containing the drying point of the standard facing upwards. The matrix solution prepared in step (6) was sprayed into the spray chamber in 24 sprays, using 1.5 mL of solution each time. The final spray amount of DCTB was 0.15 mg / cm³. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide with the side containing the drying point of the standard sample facing up in a vacuum desiccator and dry at room temperature for 15 minutes.

[0099] (10) Using a Bruker UltraFlex Ⅲ MALDI-TOF / TOF mass spectrometer, in positive ion and reflection mode, with a mass-to-charge ratio detection range of 140 to 1000, mass spectrometry analysis was performed on one side of an ITO slide containing the drying point of the standard.

[0100] (11) Based on the cleavage reaction formula of cholesterol derivatization products in the MALDI source ( Figure 6 The mass spectrometry signals of the derivatized products were extracted using Bruker DataAnalysis software, with m / z values ​​of 136.1, 180.1, and 548.5, respectively. The results are as follows: Figure 7 As shown, when DCTB is used as the matrix and 1-(carboxymethyl)-2,4,6-trimethylpyridinium is used as the derivatization reagent, mass spectrometry signals of cholesterol derivatization products on longitudinal sections of mouse brain tissue can be obtained. The distribution of cholesterol on sagittal sections of mouse brain tissue can be characterized by in-source fragmentation imaging of cholesterol derivatization products at m / z 136.1. Cholesterol is most abundant in the brainstem.

[0101] Example 6: Derivatization and MALDI mass spectrometry analysis of cholesterol in coronal sections of SOD1-G93A mouse brain tissue

[0102] (1) Take the complete brain of the SOD1-G93A model mouse frozen at -80℃, and prepare a 14µm thick sagittal section of brain tissue using a slicer. Transfer the prepared brain tissue section onto an indium tin oxide (ITO) glass slide and dry it in a vacuum desiccator at room temperature for 15 minutes.

[0103] (2) Accurately weigh 10.5 mg of 2,4,6-trimethylpyranium tetrafluoroborate, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain 2,4,6-trimethylpyranium tetrafluoroborate (10 mM) solution for later use;

[0104] (3) Accurately weigh 3.75 mg of glycine, add 5 mL of acetonitrile-water (60:40, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain glycine (10 mM) solution for later use;

[0105] (4) Mix 1 mL of the 2,4,6-trimethylpyranium tetrafluoroborate solution prepared in step (2) with 1 mL of the glycine solution prepared in step (3), and add them to 18 mL of 60% acetonitrile (ACN / H2O=6:4). Vortex to mix, and add triethylamine (1 mmol). Stir at room temperature for 10 min, then add acetic acid (2 mmol) and continue the reaction for 2 h. Freeze-dry the reaction mixture and redissolve it in DCM. Centrifuge to remove insoluble matter and extract with diethyl ether to obtain the crude product. The crude product was separated by high performance preparative liquid chromatography (HPLC). The chromatographic column was an Agilent Poroshell 120 EC-C18 (100 mm × 3.0 mm, 2.7 μm). The mobile phase was water (containing 0.5% formic acid by volume) and acetonitrile (containing 0.5% formic acid by volume) by volume. The elution gradient was 0–8 min, 2% B–100% B. The chromatographic peak at the retention time of 1.75–2 min was collected. The eluent was freeze-dried to obtain 1-(carboxymethyl)-2,4,6-trimethylpyridinium product.

[0106] (5) Dissolve the derivatization reagent prepared in step (4) in acetonitrile to a final concentration of 2 mM, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 mM), 1-hydroxybenzotriazole (1.2 mM) and triethylamine (3.6 mM) in sequence to obtain the derivatization reagent solution.

[0107] (5) Accurately weigh 50 mg of trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malonitrile (DCTB), add 5 mL of dichloromethane:methanol (50:50, v / v) solution, vortex to mix, and sonicate for 5 minutes to obtain DCTB (10 mg / mL) solution for later use.

[0108] (6) Use the Brook ImagePrep fully automatic substrate sprayer, with a spray chamber area of ​​approximately 100 cm². 2 The mouse brain tissue sections prepared in step (1) were placed on ITO glass slides with one side of the tissue section facing upwards in the center of the spray chamber. 80% ethanol was sprayed into the spray chamber in 18 sprays, using 1 mL of solution each time, with a final spray volume of 10 µL / cm. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide in a vacuum desiccator and dry it at room temperature for 15 minutes.

[0109] (7) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2The mouse brain tissue slices prepared in step (6) were placed on ITO glass slides with one side facing up in the center of the spray chamber. The derivatization reagent solution obtained in step (5) was sprayed into the spray chamber in 18 sprays, using 1 mL of solution each time. The final spray volume was 20 nmol / cm³. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide in a vacuum desiccator and dry it at room temperature for 15 minutes.

[0110] (8) Use the Brook ImagePrep fully automatic matrix sprayer, with a spray chamber area of ​​approximately 100 cm². 2 The mouse brain tissue slices prepared in step (7) were placed in the center of the spray chamber with the side of the ITO slide containing the drying point of the standard facing upwards. The matrix solution prepared in step (5) was sprayed into the spray chamber in 24 sprays, using 1.5 mL of solution each time. The final spray amount of DCTB was 0.15 mg / cm³. 2 After each layer of spraying, allow it to air dry at room temperature. After the last layer of spraying, place the ITO glass slide with the side containing the drying point of the standard sample facing up in a vacuum desiccator and dry at room temperature for 15 minutes.

[0111] (9) Using a Bruker UltraFlex Ⅲ MALDI-TOF / TOF mass spectrometer, in positive ion and reflection mode, with a mass-to-charge ratio detection range of 140 to 1000, mass spectrometry analysis was performed on one side of an ITO slide containing the drying point of the standard.

[0112] (10) Based on the cleavage reaction formula of cholesterol derivatization products in the MALDI source ( Figure 6 The mass spectrometry signal of the derivatized product was extracted using Bruker DataAnalysis software, with m / z values ​​of 136.1. The results are as follows: Figure 8 As shown, when CHCA is used as the matrix and 1-(carboxymethyl)-2,4,6-trimethylpyridinium is used as the derivatization reagent, mass spectrometry signals of cholesterol derivatization products on the coronal sections of mouse brain tissue can be obtained. The cholesterol content in the brain tissue sections of SOD1-G93A mice is higher than that in the control group and increases with age.

Claims

1. A mass spectrometry imaging method for detecting cholesterol in tissues, characterized in that, The method includes at least: 1) The derivatizing reagent carboxymethylpyridinium was synthesized in solution. The structural formula of the derivatizing reagent is as follows: ； R1 to R5 are each independently hydrogen or one, two, or three of the substituents methyl, phenyl, and benzyl, and the number of substituents is 0, one, two, three, four, or five. 2) Spray the extraction solvent onto the surface of the tissue section attached to the ITO slide, let it air dry, and then extract cholesterol. 3) Prepare a derivatization system solution by mixing the derivatization reagent and catalyst, spray it onto the surface of tissue sections, let it dry naturally, and carry out the derivatization reaction. After the reaction is completed, cholesterol derivatization products are obtained. 4) Spray the above-derivatized tissue sections with a matrix to obtain tissue section samples that can be used for mass spectrometry imaging; 5) Mass spectrometry signals of cholesterol derivatization products on tissue sections were acquired using a laser desorption / sorption ionization source (MALDI) to qualitatively identify cholesterol in the tissue and extract the mass-to-charge ratio information of the derivatization products to obtain mass spectrometry images.

2. The mass spectrometry imaging method for detecting cholesterol in tissues as described in claim 1, characterized in that, The synthesis process of the derivatization reagent is as follows: a pyranium salt solution and an amino acid solution are mixed to obtain a mixed solution, an organic base is added for catalysis, the mixture is thoroughly mixed, an organic acid is added, and the mixture is stirred at room temperature to obtain a carboxymethylpyridinium solution of the derivatization reagent. The pyranium salt wherein R1 to R5 are independently hydrogen and one, two, or three of the substituents methyl, phenyl, or benzyl, and the number of substituents is 0, one, two, three, four, or five, and the anion is BF4; its specific structural formula is (II). （Ⅱ）； The amino acid is one or more of glycine, alanine, and γ-aminobutyric acid; The concentration of the pyranium salt solution is 0.5–5 mM, and the concentration of the amino acid solution is 0.5–5 mM; the molar ratio of pyranium salt to amino acid in the mixed synthesis system solution is 10:1 to 1:

1.

3. The mass spectrometry imaging method for detecting cholesterol in tissues as described in claim 2, wherein the solvent for the pyranium salt and amino acids is an aqueous solution of acetonitrile, and the volume concentration of acetonitrile in the aqueous solution is 25-80%.

4. The mass spectrometry imaging method for detecting cholesterol in tissues as described in claim 2, characterized in that, The organic base catalyst is one or two of triethylamine or N,N-diisopropylethylamine, and the final concentration of the organic base in the synthesis system is 0.1-1 mM; the organic acid is acetic acid, and the final concentration of acetic acid in the synthesis system is 0.2-2 mM.

5. The mass spectrometry imaging method for detecting cholesterol in tissues as described in claim 1, characterized in that, The extraction solvent for cholesterol on the surface of the tissue sections is one, two, or three of the aqueous solutions of acetonitrile, methanol, and ethanol, with a volume concentration of 60-80%, and the spraying amount on the tissue section surface is 5-30 µL / cm. 2 .

6. The mass spectrometry imaging method for detecting cholesterol in tissues as described in claim 1, characterized in that, The derivatization system solution is prepared by mixing a derivatization reagent solution with a catalyst 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) solution, a 1-hydroxybenzotriazole (HOBt) solution, and triethylamine (TEA), and directly spraying the mixture onto the surface of tissue sections without incubation, allowing it to air dry. The final concentration of the derivatization reagent in the system solution is 0.5–5 mM; the final concentration of the catalyst EDCl is 0.5–5 mM; the final concentration of the catalyst HOBt is 0.5–5 mM; and the final concentration of the catalyst TEA is 1–8 mM. The solvent for the derivatization reagent, EDCl, and HOBt in the system is acetonitrile.

7. The mass spectrometry imaging method for detecting cholesterol in tissues as described in claim 1, characterized in that, The spraying amount of the derivatization system solution on one side of the tissue section is 10–100 nmol / cm². 2 .

8. The mass spectrometry imaging method for detecting cholesterol in tissues as described in claim 1, characterized in that, The matrix solution is α-cyano-4-hydroxycinnamic acid (CHCA) with a mass concentration of 5–10 mg / mL. The solvent is an acetonitrile aqueous solution with a volume concentration of 40–60%, containing trifluoroacetic acid at a volume concentration of 0.1–0.2%. The amount of CHCA matrix solution sprayed onto one side of the tissue section is 0.07–0.35 mg / cm². 2 Alternatively, the matrix solution may be trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malonitrile (DCTB) with a mass concentration of 7–35 mg / mL, and the solvent may be dichloromethane or a mixture of dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 100–50:0–50. The amount of DCTB matrix solution sprayed onto one side of the tissue section is 0.1–0.5 mg / cm². 2 .

9. The mass spectrometry imaging method for detecting cholesterol 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.

Images