An amino-reactive matrix, its method of preparation and use in mass spectrometry imaging

Abstract

The application relates to an amino reactive matrix, a preparation method thereof and application thereof in mass spectrometry imaging, and belongs to the field of organic synthesis and spatial metabolomics research. The amino reactive matrix is applied to the field of spatial omics research of metabolic small molecules of biological tissue samples, and provides important technical support for realizing spatial distribution information of amino small molecules (such as neurotransmitters, amino acids and the like) in disease model tissue samples.

Description

Technical Field

[0001] This invention relates to an amino-reactive matrix, its preparation method, and its application in mass spectrometry imaging. Background Technology

[0002] Information on the types, content, and spatial distribution of molecules in biological samples is closely related to the potential molecular mechanisms of disease origin, classification, and metastasis. Endogenous biomolecules mediate a series of life activities and play an important role in the occurrence and development of diseases. Accurate detection of metabolic molecules in clinical tissue samples is of great importance for understanding the molecular mechanisms of biological functions, promoting medical diagnosis, and providing important technical support for clinical treatment and prognosis (Trends in Neurosciences, 1993, 16: 233-240; Trends in Neurosciences, 2006, 29(8): 474-480; Neuron, 2014, 84(4): 697-707).

[0003] Biological samples are complex and diverse, ranging from small molecules such as inorganic salts, metabolites, liposomes, and peptides to large molecules such as proteins, with biomolecule concentrations spanning multiple orders of magnitude from pM to mM. Among numerous analytical methods, mass spectrometry (MS) stands out for its high resolution, high accuracy, and high sensitivity, making it particularly suitable for the detection and analysis of metabolic molecules in complex biological samples. In biomolecule detection, MS typically provides abundant qualitative and quantitative information with relatively small sample consumption, gradually evolving into a core tool for molecular recognition. With the continuous development of MS, especially the emergence of electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI) soft ionization techniques, MS is increasingly able to meet the requirements for high coverage and in-situ spatial analysis of small metabolic molecules in complex biological samples.

[0004] MALDI-MS can rapidly analyze molecular information in complex biological samples with high detection accuracy and sensitivity, and typically produces single-charged ions. Compared to ESI-MS, MALDI-MS has advantages such as speed, simplicity, and ease of automation. It is more tolerant to salt solutions and buffer solutions and is suitable for detecting the content and spatial distribution of low concentrations of metabolic molecules in tissue sections. Therefore, it is more widely used in mass spectrometry imaging and other analyses (Nature Reviews Neurology, 2015, 11(10): 567-584; Molecules, 2023, 28(14): 5373).

[0005] Mass spectrometry imaging (MSI) is a molecular imaging technique that uses a mass spectrometer and its accompanying imaging control software to analyze the tissue surface point by point, collecting mass spectra at various coordinates and plotting a signal intensity map over the entire sample area to create an ion image. Compared to other imaging methods, this method has advantages such as label-free imaging, simultaneous imaging of a large number of molecules, good molecular specificity, and high spatial resolution. The workflow of mass spectrometry imaging includes four steps: first, acquiring and preparing the sample to be detected; second, defining the surface area to be used for imaging the sample; third, acquiring the mass-to-charge ratio and ion intensity map of each pixel point; finally, data processing, combining the signal intensity of the ions and their position on the sample surface to draw a two-dimensional distribution map of the ions on the sample surface; further data processing to obtain a three-dimensional spatial distribution map of the analyte in the sample, and more intuitively analyzing the relationship between the spatial distribution of small molecules and diseases (Neuron, 2014, 84(4): 697-707; Nature Methods, 2019, 16(10): 1021-1028; Analytical Chemistry, 2018, 90(22): 13580-13590; Trends in Analytical Chemistry, 2022, 157: 116809).

[0006] In MALDI imaging, the matrix plays a crucial role in absorbing laser energy to aid analyte resolution and ionization. The generally accepted mechanism of matrix action is as follows: the matrix co-crystallizes with the sample, absorbs the laser, and then transfers the energy to the analyte molecules, promoting their ionization and volatilization before entering mass spectrometry analysis. Mass spectrometry is an in-situ detection and analysis method for tissue samples, which cannot perform chromatographic separation, posing significant challenges to the selectivity and high sensitivity of target analytes. The matrix greatly influences the effectiveness of mass spectrometry imaging, including: mass spectrometry sensitivity (the physicochemical properties of the matrix directly affect the ionization efficiency of the analyte); selectivity (tissue composition is complex, and analyte molecules are easily interfered with and suppressed; a well-designed and synthesized matrix helps improve analytical selectivity); accuracy (the uniformity of matrix coverage within the tissue and the matrix solvent can cause small molecule delocalization, significantly affecting the accuracy of spatial molecular distribution imaging); and resolution (during sample preparation, the matrix spraying process may generate large matrix crystal particles, reducing the resolution of mass spectrometry imaging). Therefore, a well-designed matrix is ​​helpful for mass spectrometry imaging analysis (Frontiers in Endocrinology, 2023, 13: 993081; Analytical and Bioanalytical Chemistry, 2020, 413(10): 2599-2617).

[0007] In the process of mass spectrometry imaging analysis, some molecules have problems such as low ionization efficiency, ion suppression, low concentration, and poor molecular stability, making it difficult for traditional mass spectrometry imaging technology to directly detect them. Chemical derivatization can solve the above problems better. It is a common mass spectrometry sensitization method. It can achieve mass spectrometry sensitization detection of biomolecules by introducing various modified groups. For example, for the functional groups on the analyte molecule, a derivatization reagent is designed to react with it and introduce functional groups with high ionization efficiency. The derivatization product will further improve the ionization efficiency of the molecule; improve the selectivity of the molecule; increase the molecular weight and reduce background interference; improve the stability of the molecule, and can be used to detect some volatile substances (Nature Methods, 2019, 16(10): 1021-1028; Analytical Chemistry, 2018, 90(22): 13580-13590; Trends in Analytical Chemistry, 2022, 157: 116809).

[0008] For the study of the spatial distribution of amino small molecules in brain tissue, derivatization mass spectrometry imaging technology can significantly improve the sensitivity and molecular stability of mass spectrometry detection. For example, Kaya et al. developed an N-methylpyridinylphenylboronic acid derivatization matrix, which derivatized dopamine, norepinephrine, and epinephrine through the affinity cyclization reaction of phenylboronic acid with cis-dihydroxyl groups (Analytical Chemistry, 2018, 90(22):13580-13590); the matrix framework uses N-methylpyridinium positive ion groups, which improves the ionization efficiency of the analytes. However, due to the limitations of the derivatization reaction itself, this technique can only analyze catecholamines, and the analytical throughput is low; in addition, the derivatization reaction requires alkaline conditions, which has poor biocompatibility with tissue samples. To improve the analytical throughput of neurotransmitters, Shariatgorji et al. developed a 2,4-diphenyl-pyranyltetrafluoroborate (DPP-TFB) derivatization matrix based on the reaction of pyran salts with primary amines. This matrix can recognize various neurotransmitters such as dopamine, serotonin, 4-aminobutyric acid (GABA), and glutamate. However, this reaction still requires relatively alkaline conditions, and the reaction needs to be quenched with acetic acid after the derivatization reaction, making the operation steps rather cumbersome. Furthermore, this reaction cannot recognize neurotransmitters containing secondary amine groups (Neuron, 2014, 84(4):697-707). To improve the biocompatibility of the derivatization reaction, the research group proposed a fluoromethylpyridinium salt derivatization matrix (FMP) to achieve covalent labeling of neurotransmitters and their related metabolites containing phenolic hydroxyl groups or primary and secondary amine groups, thereby improving the detection limit of MALDI mass spectrometry for various low-abundance neurotransmitters. However, the derivatization reaction is inefficient (requires 3 hours), the matrix spraying process requires a high temperature (80℃), and long-term spraying may cause spatial delocalization of the analyte (Nature Methods, 2019, 16(10): 1021-1028).

[0009] In summary, although amino-based small molecule mass spectrometry imaging technology based on derivatized matrices is effective, it still has some limitations, such as low reaction efficiency, harsh reaction conditions, and poor biocompatibility, which lead to low detection sensitivity and coverage of amino-based small molecules and may produce false positive results. Summary of the Invention

[0010] This invention relates to an organic molecule that is a multifunctional matrix with the following functional characteristics: it contains a succinimide ester reactive group, which undergoes amidation reactions with amino-based small molecules under physiological conditions, resulting in mild and efficient reactions; it contains a pyridinium ion group, which imparts a stable positive charge to the amino-based small molecules after derivatization, enhancing their signal intensity during mass spectrometry imaging; it contains anthracene pyridine structural units, improving its laser absorption capacity and aiding in the resolution and ionization of amino-based small molecules; it contains bromine or chlorine elements, which impart a pair of characteristic mass spectrometry ion peaks to the amino-based small molecules after derivatization, improving identification accuracy by extracting these characteristic ion peaks; and it possesses good crystallinity, eliminating the need for additional matrix addition during mass spectrometry imaging, thus simplifying the imaging process. This amino-reactive matrix is ​​applied in the field of spatial omics research on metabolic small molecules in biological tissue samples, providing crucial technical support for realizing the spatial distribution information of amino-based small molecules (such as neurotransmitters and amino acids) in disease model tissue samples.

[0011] Based on the current research status and design principles of matrices in mass spectrometry imaging, this invention designs and synthesizes an amino-reactive matrix (Examples 1 and 2). Figure 1 (2, 3, and 4). The organic molecule of this invention is a multifunctional matrix with the following functional characteristics: it possesses a succinimide ester reactive group, undergoes amidation reaction with amino small molecules under physiological conditions, and the reaction conditions are mild and efficient. Figure 3 and 4 ); possessing pyridinium ion groups, the amino-based small molecules acquire a stable positive charge after derivatization, thus enhancing their signal intensity during mass spectrometry imaging (Examples 3 and 4, Figure 8 and 9 It contains anthracene pyridine structural units, which enhance its laser absorption capacity and assist in the resolution and ionization of amino-based small molecules; it contains bromine or chlorine elements, and after derivatization, amino-based small molecules yield a pair of characteristic mass spectrometry ion peaks. By extracting these characteristic ion peaks, the accuracy of their identification is improved. Figure 5 , 6 (and 8); it has good crystallinity and does not require the addition of an additional matrix during mass spectrometry imaging, thus simplifying the operation steps of mass spectrometry imaging (Examples 3 and 4). The amino-reactive matrix of the present invention is applied to the field of spatial omics research on small metabolic molecules in biological tissue samples, providing important technical support for realizing the spatial distribution information of amino-based small molecules (such as neurotransmitters, amino acids, etc.) in disease model tissue samples. Figure 7 , 9 10 and 11).

[0012] The amino-reactive matrix provided by this invention has the following structure:

[0013]

[0014] Where X is a substituent, one or both of the elements bromine or chlorine.

[0015] Specifically, it includes two structures, as shown in the following formula:

[0016] Structure of bromine-containing amino reactive matrix:

[0017]

[0018] Structure of chlorine-containing amino reactive matrix:

[0019]

[0020] This invention provides a method for preparing an amino-reactive matrix, the specific steps of which are as follows:

[0021] In the first step, reactants 9,10-dibromo(or chloro)anthracene (compound 1), 4-pyridinephenylboronic acid, tetrakis(triphenylphosphine)palladium, and sodium carbonate are dissolved in toluene, with the molar ratio of the four reagents controlled at 1:1.

[0022] (1.0-1.5):(5%-10%):(4.0-5.0), the reaction temperature was controlled at 100-110℃, and the reaction time was controlled at 12-24h; after the reaction was completed, the crude product was separated and purified by column chromatography. The separation packing material was 200-400 mesh silica gel, the mobile phase was a methanol-chloroform mixture, and the volume ratio of methanol to chloroform was controlled at 1:(4.0-5.0). After removing the organic phase, a pale yellow solid (compound 2) was obtained.

[0023] In the second step, compound 2 and 3-iodopropionic acid are dissolved in acetonitrile, with the molar ratio of compound 2 to 3-iodopropionic acid controlled at 1:(2.5-3.5). The reaction temperature is controlled at 120-135℃, and the reaction time is controlled at 12-36h. After the reaction is completed, the reaction solution is purified by semi-preparative liquid phase separation. The mobile phases are water (containing 0.1-0.5% TFA by volume) and acetonitrile (containing 0.1-0.5% TFA by volume), respectively. A linear gradient is used: the aqueous phase is increased from 2-4% to 40-60% in 30 min. The eluent after 24-28 min is collected, and the solvent is removed by rotary evaporation under reduced pressure to obtain a pale yellow solid 9-bromo(or chloro)-10-anthrapyridinylpropionic acid iodate (compound 3).

[0024] In the third step, compound 3, EDC, and NHS are dissolved in DMSO. The molar ratio of sebacic acid, EDC, and NHS is controlled at 1:(1.0-1.5):(1.0-1.5). The reaction temperature is controlled at 25-30℃, and the reaction time is controlled at 12-24h. After the reaction is completed, the reaction solution is purified by semi-preparative liquid phase separation. The mobile phases are water (containing 0.1-0.5% TFA by volume) and acetonitrile (containing 0.1-0.5% TFA by volume). A linear gradient is used: the aqueous phase is increased from 2-4% to 60-80% in 40 min. The eluent after 32-35 min is collected and freeze-dried under vacuum to obtain 9-bromo(or chloro)-10-anthrapyridinylpropionate succinimide iodate (compound 4, i.e., the target matrix reagent).

[0025]

[0026] The amino-reactive matrix of this invention can be used in the field of spatial omics research on small metabolic molecules in biological tissue samples, providing important technical support for realizing the spatial distribution information of amino-based small molecules (such as neurotransmitters, amino acids, etc.) in disease model tissue samples.

[0027] Compared with existing matrices, the amino-reactive matrix of the present invention has the following advantages:

[0028] 1) It has a succinimide ester reactive group, which can undergo amidation reaction with amino small molecules under physiological conditions. The reaction conditions are mild and efficient.

[0029] 2) It has a pyridinium ion group, and after derivatization, the amino-based small molecules have a stable positive charge, which improves their signal intensity in the mass spectrometry imaging process;

[0030] 3) It has anthracene pyridine structural units, which enhance its laser absorption capacity and assist in the desorption and ionization of amino-based small molecules;

[0031] 4) When amino-based small molecules containing bromine or chlorine undergo derivatization, they produce a pair of characteristic mass spectrometry ion peaks. By extracting these characteristic ion peaks, the accuracy of their identification can be improved.

[0032] 5) It has good crystallization properties and does not require the addition of an additional matrix during mass spectrometry imaging, thus simplifying the operation steps of mass spectrometry imaging. Attached Figure Description

[0033] Figure 1 The general structural formula for amino-reactive matrices;

[0034] Figure 2 Synthetic routes for amino-reactive matrices;

[0035] Figure 3 The specific structural formula of the bromine-containing amino reactive matrix;

[0036] Figure 4 The specific structural formula of the chlorine-containing amino reactive matrix;

[0037] Figure 5 Characterization images of bromine-containing amino reactive matrices by liquid chromatography, 1H NMR, 1C NMR and high-resolution mass spectrometry.

[0038] Figure 6 Characterization of chlorine-containing amino reactive matrix by liquid chromatography, proton NMR, carbon NMR and high-resolution mass spectrometry;

[0039] Figure 7 A technical roadmap for the application of amino-reactive matrices in mass spectrometry imaging;

[0040] Figure 8 Mass spectra of characteristic ions after derivatization from a bromide-containing amino reactive matrix;

[0041] Figure 9 Spectra of metabolic substances detected in bromine-containing amino reactive matrix;

[0042] Figure 10 Spatial distribution map of metabolites detected by bromine-containing amino reactive matrix;

[0043] Figure 11 Spatial distribution of dopamine molecules detected in a bromine-containing amino reactive matrix in the mouse brain. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments.

[0045] Example 1

[0046] This embodiment discloses a method for preparing a bromine-containing amino reactive matrix, comprising three reaction steps, as shown below:

[0047] Step 1: Preparation of 9-bromo-10-pyridinylanthracene. 9,10-Dibromoanthracene (1.01 g, 3 mmol), 4-pyridinephenylboronic acid (443 mg, 3.6 mmol), tetrakis(triphenylphosphine)palladium (173 mg, 0.15 mol), and sodium carbonate (1.59 g, 15 mmol) were dissolved in 50 mL of toluene and refluxed at 110 °C for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by column chromatography using 200-400 mesh silica gel as the packing material and a methanol-chloroform mixture (methanol-chloroform volume ratio 1:3) as the mobile phase. The organic phase was removed by rotary evaporation under reduced pressure, yielding 801.6 mg of a pale yellow solid (compound 2-1), with a yield of 80%. 1¹H NMR (400MHz, DMSO-d6, ppm): δ 8.12 (s, 1H), 7.72 (s, 2H), 7.63 (s, 2H), 7.36 (s, 1H); HR-MS (C19H12BrN): Theoretical value: 333.0153, Measured value [M+H] + 334.0231.

[0048] The second step involved the preparation of 9-bromo-10-anthracenepyridinylpropionic acid iodate. Compound 2-1 (666 mg, 2 mmol) and 3-iodopropionic acid (600 mg, 6 mmol) were dissolved in 100 mL of anhydrous acetonitrile and refluxed at 135 °C for 36 h. After the reaction was complete, the reaction solution was purified by semi-preparative liquid chromatography. The mobile phases were water (containing 0.1% TFA by volume) and acetonitrile (containing 0.1% TFA by volume), using a linear gradient: increasing the aqueous phase from 2% to 60% over 30 min. The eluent was collected after 24–28 min, and the solvent was removed by rotary evaporation under reduced pressure to obtain 801 mg of the pale yellow solid 9-bromo-10-anthracenepyridinylpropionic acid iodate (compound 3-1), with a yield of 75%. 1 H NMR (400MHz, DMSO-d6, ppm) δ8.12 (s, 1H), 7.72 (s, 2H), 7.63 (s, 2H), 7.36 (s, 1H), 3.82 (t, d, J = 6.4Hz, 2H), 2.56 (t, d, J = 6.4Hz, 2H); HR-MS (C22H17BrNO2 + Theoretical value: 406.0436, Measured value [M] + 406.0439.

[0049] The third step involved the preparation of 9-bromo-10-anthracenepyridinylpropionate succinimide iodate (compound 4-1, the target matrix reagent). Compound 3-1 (533 mg, 1 mmol), EDC (191.7 mg, 1 mmol), and NHS (115 mg, 1 mmol) were dissolved in 30 mL of anhydrous DMSO and reacted at room temperature (25 °C) for 12 h. After the reaction was complete, the reaction solution was purified by semi-preparative liquid chromatography. The mobile phases were water (containing 0.1% TFA by volume) and acetonitrile (containing 0.1% TFA by volume), using a linear gradient: increasing the aqueous phase from 2% to 80% over 40 min. The eluent was collected after 32-35 min and lyophilized under vacuum to obtain 504 mg of 9-bromo-10-anthracenepyridinylpropionate succinimide iodate (compound 4-1, the target matrix reagent), with a yield of 80%. 1H NMR (400MHz, DMSO-d6, ppm) δ8.12(s,1H),7.72(s,2H),7.63(s,2H),7.36(s,1H),3.82(t,d,J=6.4Hz,2H),2.81(s,4H),2.56(t,d,J=6.4Hz,2H); 1 C NMR (400MHz, DMSO-d6, ppm) δ169.0,167.4,152.5,146.4,137.7,131.2,127.4,126.1,125.8,123.9,50.3,29.3,25.6; HR-MS(C26H20BrN2O4 + Theoretical value: 503.0601, Measured value [M] + :503.0609.

[0050]

[0051] Example 2

[0052] This embodiment discloses a method for preparing a chlorine-containing amino reactive matrix, comprising three reaction steps, as shown below:

[0053] Step 1: Preparation of 9-chloro-10-pyridinylanthracene. 9,10-Dichloroanthracene (741 mg, 3 mmol), 4-pyridinephenylboronic acid (443 mg, 3.6 mmol), tetrakis(triphenylphosphine)palladium (173 mg, 0.15 mol), and sodium carbonate (1.59 g, 15 mmol) were dissolved in 50 mL of toluene and refluxed at 110 °C for 20 h. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by column chromatography using 200-400 mesh silica gel as the packing material and a methanol-chloroform mixture (methanol-chloroform volume ratio 1:5) as the mobile phase. The organic phase was removed by rotary evaporation under reduced pressure, yielding 739.5 mg of a pale yellow solid (compound 2-2), with a yield of 85%. 1 ¹H NMR (400MHz, DMSO-d⁶, ppm): δ 8.10 (s, 1H), 7.75 (s, 2H), 7.59 (s, 2H), 7.41 (s, 1H); HR-MS (C₁₉H₁₂ClN): Theoretical value: 289.0658, Measured value [M+H] + 290.0737.

[0054] The second step involved the preparation of 9-chloro-10-anthracenepyridinylpropionic acid iodate. Compound 2-2 (578 mg, 2 mmol) and 3-iodopropionic acid (600 mg, 6 mmol) were dissolved in 100 mL of anhydrous acetonitrile and refluxed at 130 °C for 24 h. After the reaction was complete, the reaction solution was purified by semi-preparative liquid chromatography. The mobile phases were water (containing 0.1% TFA by volume) and acetonitrile (containing 0.1% TFA by volume), using a linear gradient: increasing the aqueous phase from 2% to 60% over 30 min. The eluent was collected after 24-26 min, and the solvent was removed by rotary evaporation under reduced pressure to obtain 735 mg of the pale yellow solid 9-chloro-10-anthracenepyridinylpropionic acid iodate (compound 3-3), with a yield of 75%. 1 H NMR (400MHz, DMSO-d6, ppm) δ8.16 (s, 1H), 7.69 (s, 2H), 7.59 (s, 2H), 7.28 (s, 1H), 3.79 (t, d, J = 6.4Hz, 2H), 2.49 (t, d, J = 6.4Hz, 2H); HR-MS (C22H17ClNO2 + Theoretical value: 362.0942, Measured value [M] + 362.0949.

[0055] The third step involved the preparation of 9-chloro-10-anthracenepyridinylpropionate succinimide iodate (compound 4-2, the target matrix reagent). Compound 3-2 (363 mg, 1 mmol), EDC (191.7 mg, 1 mmol), and NHS (115 mg, 1 mmol) were dissolved in 30 mL of anhydrous DMSO and reacted at room temperature (25 °C) for 12 h. After the reaction was complete, the reaction solution was purified by semi-preparative liquid chromatography. The mobile phases were water (containing 0.1% TFA by volume) and acetonitrile (containing 0.1% TFA by volume), using a linear gradient: increasing the aqueous phase from 2% to 80% over 40 min. The eluent was collected after 33-35 min and lyophilized under vacuum to obtain 498.1 mg of 9-chloro-10-anthracenepyridinylpropionate succinimide iodate (compound 4-2, the target matrix reagent), with a yield of 85%. 1 H NMR (400MHz, DMSO-d6, ppm) δ8.17(s,1H),7.68(s,2H),7.57(s,2H),7.46(s,1H),3.72(t,d,J=6.2Hz,2H),2.83(s,4H),2.54(t,d,J=6.4Hz,2H); 1C NMR (400MHz, DMSO-d6, ppm) δ169.7,166.4,154.5,148.5,136.4,136.5,126.9,124.5,123.3,123.1,48.5,28.9,24.8; HR-MS(C26H20ClN2O4 + Theoretical value: 459.1106, Measured value [M] + :459.1110.

[0056]

[0057] Example 3

[0058] The bromine-containing amino-reactive matrix from Example 1 was applied to mouse brain mass spectrometry imaging for spatial omics analysis of neurotransmitter molecules. The specific steps are as follows:

[0059] Fresh mouse brains (ethical review pending, with animal testing qualifications) were harvested and sliced ​​into 10 μm thick sections using a cryostat. A bromine-containing amino-reactive matrix with a concentration of 4.0 mmol was prepared. 30.8 mg of the matrix was dissolved in 5 mL of a 60% acetonitrile / water solution. The matrix was then uniformly sprayed onto the surface of the mouse brain sections using a matrix sprayer at a flow rate of 0.15 mL / min, a spraying temperature of 25 °C, and dried for 20 min. The spraying volume was 0.2 mL / cm². 2 The sample was placed in an imaging mass spectrometry microscope. A mouse brain scanning region was selected, with a resolution of 10 μm, a mass-to-charge ratio (m / z) of 100-1500, a laser wavelength of 355 nm, and an acquisition frequency of 2000 Hz, using positive ion mode. After mass spectrometry data acquisition, characteristic ion peaks were extracted (signal intensities of characteristic ion peaks differing by 2 Da are 1:1). The structure of amino small molecules was determined by searching a metabolic database, and their spatial distribution in the mouse brain slices was plotted based on changes in ion intensity. See [link to detailed steps] for more information. Figure 7 For details, please see Figure 9 and Figure 10 The results showed that small metabolic molecules such as dopamine are mainly distributed in brain regions such as the cerebral cortex and striatum. This distribution pattern is consistent with the distribution pattern of nerve cells in the brain, indicating that the life activities in this region are more vigorous compared to other regions.

[0060] Example 4

[0061] The chlorine-containing amino-reactive matrix from Example 2 was applied to mouse brain mass spectrometry imaging for spatial omics analysis of neurotransmitter molecules. The specific steps are as follows:

[0062] Fresh mouse brains (ethical review pending, with animal testing qualifications) were harvested and sliced ​​to a thickness of 15 μm using a cryostat. A bromine-containing amino-reactive matrix with a concentration of 4.0 mmol was prepared. 27.4 mg of the matrix was dissolved in 5 mL of a 60% acetonitrile / water solution. The matrix was then uniformly sprayed onto the surface of the mouse brain slices using a matrix sprayer at a flow rate of 0.2 mL / min, a spraying temperature of 28 °C, and dried for 30 min. The spraying volume was 0.2 mL / cm³. 2 The sample was placed in an imaging mass spectrometry microscope. A mouse brain scanning region was selected, with a resolution of 20 μm, a mass-to-charge ratio (m / z) of 100-1500, a laser wavelength of 355 nm, and an acquisition frequency of 1500 Hz, using positive ion mode. After mass spectrometry data acquisition, characteristic ion peaks (signal intensities differing by 2 Da were 3:1) were extracted. The structure of amino small molecules was determined by searching a metabolic database, and their spatial distribution in the mouse brain slices was plotted based on changes in ion intensity. The results showed that dopamine and other metabolic small molecules were mainly distributed in brain regions such as the cerebral cortex and striatum. This distribution pattern is consistent with the distribution pattern of nerve cells in the brain, indicating that the life activity in this region is more vigorous compared to other regions.

Claims

1. An amino-reactive matrix, the chemical structural formula of which is: in, X is a substituent, which can be one or both of the elements bromine or chlorine.

2. A method for preparing the matrix according to claim 1, characterized in that, The specific process is as follows: Step 1: 9,10-dibromo (and / or 9,10-dichloro)anthracene (compound 1) undergoes the Suzuki reaction with 4-pyridinephenylboronic acid, with toluene as the reaction solvent, tetrakis(triphenylphosphine)palladium as the catalyst, and sodium carbonate as the base additive, to prepare 9-bromo (and / or chloro)-10-pyridineanthracene (compound 2). Step 2: Compound 2 undergoes an electrophilic substitution reaction with 3-iodopropionic acid, using acetonitrile as the reaction solvent, to prepare 9-bromo(and / or chloro)-10-anthrapyridylpropionic acid iodate (compound 3); Step 3: Compound 3 undergoes an esterification reaction with N-hydroxysuccinimide ester (NHS), using dimethyl sulfoxide (DMSO) as the reaction solvent and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as the coupling agent to prepare 9-bromo(and / or chloro)-10-anthrapyridylpropionate succinimide iodate (compound 4, i.e., the target matrix reagent).

3. The method for preparing the matrix according to claim 2, characterized in that: In step one, reactants 9,10-dibromo(and / or chloro)anthracene (compound 1), 4-pyridinebenzolic acid, tetrakis(triphenylphosphine)palladium, and sodium carbonate are dissolved in toluene. The molar ratio of the four reagents is controlled at 1:(1.0-1.5):(5%-10%):(4.0-5.0). The reaction temperature is controlled at 100-110℃, and the reaction time is controlled at 12-24h. After the reaction is completed, the crude product is separated and purified by column chromatography. The separation packing material is 200-400 mesh silica gel, and the mobile phase is a methanol-chloroform mixture. The volume ratio of methanol to chloroform is controlled at 1:(4.0-5.0). After removing the organic phase, a pale yellow solid (compound 2) is obtained.

4. The method for preparing the matrix according to claim 2, characterized in that: In step two, compound 2 and 3-iodopropionic acid are dissolved in acetonitrile, with the molar ratio of compound 2 to 3-iodopropionic acid controlled at 1:(2.5-3.5). The reaction temperature is controlled at 120-135℃, and the reaction time is controlled at 12-36h. After the reaction is completed, the reaction solution is purified by semi-preparative liquid phase separation. The mobile phases are water (containing 0.1-0.5% TFA by volume) and acetonitrile (containing 0.1-0.5% TFA by volume), respectively. A linear gradient is used: the aqueous phase is increased from 2-4% to 40-60% in 30 min. The eluent after 24-28 min is collected, and the solvent is removed by rotary evaporation under reduced pressure to obtain a pale yellow solid 9-bromo(and / or chloro)-10-anthrapyridylpropionic acid iodate (compound 3).

5. The method for preparing the matrix according to claim 2, characterized in that: In step three, compound 3, EDC, and NHS are dissolved in DMSO. The molar ratio of sebacic acid, EDC, and NHS is controlled at 1:(1.0-1.5):(1.0-1.5), the reaction temperature is controlled at 25-30℃, and the reaction time is controlled at 12-24h. After the reaction was completed, the reaction solution was purified by semi-preparative liquid phase separation. The mobile phases were water (containing 0.1-0.5% TFA by volume) and acetonitrile (containing 0.1-0.5% TFA by volume), respectively. A linear gradient was used: the aqueous phase was increased from 2-4% to 60-80% in 40 min. The eluent was collected after 32-35 min and freeze-dried under vacuum to obtain 9-bromo(and / or chloro)-10-anthrapyridinylpropionate succinimide iodate (compound 4, i.e., the target matrix reagent).

6. The application of the matrix according to claim 1 in metabolic molecular mass spectrometry imaging of biological tissue samples.

7. The application according to claim 6, characterized in that: The matrix described herein can be used in the field of spatial omics research on metabolic small molecules in biological tissue samples, providing important technical support for realizing the spatial distribution information of amino-based small molecules (such as one or more of neurotransmitters and amino acids) in disease model tissue samples.

8. The application according to claim 6, characterized in that: Fresh mouse brains were collected and sliced ​​to a thickness of 10-15 μm using a cryostat. A matrix concentration of 4.0-5.0 mmol was prepared by dissolving the matrix in an acetonitrile / water system, with the acetonitrile volume ratio being 40-60%. The matrix was then uniformly sprayed onto one side of the mouse brain slice using a matrix sprayer at a flow rate of 0.1-0.3 mL / min and a spraying temperature of 25-30℃. The slices were dried for 20-30 min, with a spraying volume of 0.2-0.4 mL / cm³. 2 The sample was placed in an imaging mass spectrometry microscope; the scanning area of ​​the mouse brain was selected, the resolution was set to 10-50 μm, the mass-to-charge ratio m / z was set to 100-1500, the laser wavelength was set to 355 nm, the acquisition frequency was 1500-2000 Hz, and the data was acquired in positive ion mode. After mass spectrometry data acquisition, characteristic ion peaks were extracted (the characteristic ion peaks in the bromine-containing matrix were 1:1; the characteristic ion peaks in the chloride-containing matrix were 3:1). The structure of amino small molecules was determined by searching the metabolic database, and their spatial distribution in mouse brain slices was plotted by measuring changes in ion intensity.

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