Method and analysis system for detecting spatial distribution of brain nucleic acid hydrolysate based on mass spectrum imaging

Abstract

The invention discloses a method for detecting spatial distribution of brain nucleic acid hydrolysate based on mass spectrum imaging and an analysis system.The method comprises the steps of sample preparation, mass spectrum imaging and spatial quantitative analysis of nucleic acid hydrolysate, specifically, a spray needle capillary tube with the diameter being 10-30 micrometers is adopted, the angle between a spray needle and a sample glass slide is adjusted to be 55-57 degrees, the height is 35-36.5 mm, and the sample glass slide is placed in the sample capillary tube; the method comprises the following steps: uniformly spraying a spray solvent on the surface of a brain slice, scanning the whole sample to obtain mass spectrum imaging data, extracting the ion strength of target objects corresponding to different brain regions according to the monitored ion mass-to-charge ratio of the nucleic acid hydrolysate, and obtaining the spatial distribution of the nucleic acid hydrolysate in the different brain regions of the brain; the analysis system can extract the ion strength of the target object corresponding to the different brain regions according to the obtained brain mass spectrum and the monitored ion mass-to-charge ratio of the target object, the spatial distribution of the target object in the different brain regions is obtained, and spatial difference analysis among different groups is completed.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a method and an analysis system for detecting the spatial distribution of nucleic acid hydrolysis products in the brain based on mass spectrometry imaging. Background Art

[0002] A large number of studies have shown that RNA modifications or DNA modifications are closely related to biological development and disease occurrence. Among them, RNA modifications can regulate RNA translation, change RNA stability, and post-transcriptional modifications of RNA are dynamically changing. Any misregulation of them may have a significant impact on RNA metabolism, which is crucial for cell function. Therefore, RNA modifications play a crucial role in most biological activities and mediate the normal development of the brain. Among them, N6-methyladenosine (m 6 A) is a modification widely present in mRNA, and its formation is catalyzed by a methyltransferase complex, where the mettl3 enzyme is a key factor. Research has shown that the inactivation of mettl3 in the nervous system of mice leads to the absence of m 6 A modification, an extended RNA half-life, and abnormal splicing, resulting in dysregulation of gene expression across the transcriptome. Conditional knockout of mettl3 in mice leads to abnormal shrinkage of the cerebellum and the inability to develop into cerebellar folia structures, easily causing balance disorders. It can be seen that m 6 A mediated by mettl3 plays an important role in the development of the mammalian cerebellum.

[0003] Recent studies have shown that the key cause of Alzheimer's disease may be related to disorders of RNA modifications. In the nerve cells of Alzheimer's disease patients, as misfolded tau proteins accumulate continuously, the m 6 A modification level on mRNA will increase by more than 4 times. Subsequently, with the help of the RNA-binding protein HNRNPA2B1, the RNA labeled with m 6 A will specifically bind to the misfolded tau protein, which may be related to the formation of tau protein precipitation in Alzheimer's disease. At the same time, during stress, m 6 A modification can induce phase separation in cells, sequester some mRNAs related to cell repair, and inhibit the synthesis of downstream repair proteins so that they cannot participate in cell repair. In Alzheimer's disease, the stress granules generated by the phase separation process are permanent or long-term existing, so that in this pathological state, the mRNAs encoding cell repair proteins are continuously inhibited, ultimately leading to abnormal cell metabolism and death. However, in addition to m6A, there are other modifications in mRNA. It is currently unclear whether there are other RNA modifications involved in the progression of Alzheimer's disease in addition to m 6 A modification and the spatial distribution of RNA modifications in different regions of the brain.

[0004] The nucleic acid hydrolysis products include free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides. The spatial distribution of their hydrolysis products in the brain can reflect the metabolic level of nucleic acids and the modification level before degradation in the brain region. However, the existing fluorescence or immunofluorescence imaging detection methods using antibodies to bind modified nucleosides are prone to false positive results. Although some studies have used liquid chromatography-tandem mass spectrometry to detect free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides in the mouse brain, this method grinds the whole brain to obtain brain homogenate for detection. This grinding will destroy the spatial distribution information of these nucleic acid hydrolysis products in the brain and cannot be used to detect the distribution of free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides in different brain regions. And dissecting the brain to separate different brain regions and then homogenizing for detection is very time-consuming and laborious. Moreover, the corpus callosum and fornix are commissural fibers that are difficult to separate, and it is even more difficult to be precise to brain subregions. The spatial distribution of nucleic acid hydrolysis products in the brain plays a crucial role in studying brain development and brain diseases. However, there are currently no reports on the detection of the spatial distribution of various free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides in the brain.

[0005] With the development of mass spectrometry imaging technology, by combining mass spectrometry imaging technology with professional image processing software, directly analyzing biological tissue sections, generating two-dimensional ion intensity maps of compounds with any specified mass-to-charge ratio (m / z), and performing high-throughput, comprehensive, and rapid analysis of the composition, relative abundance, and distribution of compounds in the tissue, biomarker discovery and monitoring can be carried out through the spatial distribution of the obtained potential biomarkers. However, there are thousands of metabolites in the brain. Complex lipids account for the largest proportion in the brain metabolome, while nucleosides, nucleotides, and their analogs account for about 2.4%. In addition, many endogenous metabolites may have very similar masses to the target nucleosides and nucleotides. Using existing mass spectrometry imaging technology to identify free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides with low content is difficult to accurately and quickly identify various free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides in different brain regions due to the difficulty in clearly identifying the brain subregion structure and the large difficulty in later data analysis. Therefore, studying how to improve the spatial resolution of existing mass spectrometry imaging technology for different brain regions in the brain, facilitating the accurate identification of more different brain regions, and facilitating the rapid detection of the spatial distribution of various nucleic acid hydrolysis products in the brain can provide a new research direction for studying the molecular mechanisms of certain diseases. Summary of the Invention

[0006] In view of the above deficiencies or improvement requirements of the prior art, the present invention provides a method and an analysis system for detecting the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging. The purpose is to use a spray needle capillary with a diameter of 10 to 30 microns based on mass spectrometry imaging technology, adjust the angle between the spray needle and the sample slide to 55° to 57°, and the height to 35 to 36.5 mm, detect brain slices, and the obtained mass spectrometry imaging ion map can clearly identify different brain region structures such as the corpus callosum and fornix on the sagittal plane and the dentate gyrus in the hippocampus on the coronal plane. Further, by improving the spray solvent system, the sensitivity of the existing mass spectrometry imaging for detecting nucleic acid hydrolysis products is improved, and by constructing a mass spectrometry imaging database of nucleic acid hydrolysis products, the accuracy of the existing mass spectrometry imaging for detecting nucleic acid hydrolysis products is improved, thereby solving the technical problem that the existing mass spectrometry imaging has a low spatial resolution for different sub-brain region structures of the brain and is difficult to accurately identify the sub-brain region structures of the brain.

[0007] To achieve the above object, according to one aspect of the present invention, a method for detecting the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging is provided, which includes the following steps:

[0008] (1) Sample preparation: Affix the frozen brain slices to be measured on a positively charged slide to obtain a sample slide;

[0009] (2) Mass spectrometry imaging: Use a spray needle capillary with a diameter of 10 to 30 microns, adjust the angle between the spray needle and the sample slide to 55° to 57°, and the height to 35 to 36.5 mm, evenly spray the spray solvent in the spray needle onto the surface of the brain slice, perform in-situ desorption and ionization of the metabolites on the brain slice, and select the corresponding ion channel for imaging in the negative ion acquisition mode according to whether the brain slice is sagittal or coronal to obtain mass spectrometry imaging data;

[0010] (3) Spatial quantitative analysis of nucleic acid hydrolysis products: Based on the mass spectrometry imaging data obtained in step (2), extract the ion intensities of the target substances corresponding to different brain regions according to the monitored ion mass-to-charge ratios of the nucleic acid hydrolysis products to obtain the spatial distribution of the nucleic acid hydrolysis products in different brain regions of the brain;

[0011] The nucleic acid hydrolysis products are hydrolysis products of RNA and / or DNA, including one or more of free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides.

[0012] Preferably, in the method for detecting the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging, the spray solvent is an acetonitrile-water mixed solution, where the volume ratio of acetonitrile to water is (5 to 8):(2 to 5); preferably, the volume ratio of acetonitrile to water is 8:2.

[0013] Preferably, for the method of detecting the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging, a spray needle capillary with a diameter of 20 microns is used, and the angle between the spray needle and the sample slide is adjusted to 57°, and the height is 36.5 mm for spraying.

[0014] Preferably, for the method of detecting the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging, the flow rate of the spray solvent is controlled so that the spray points are oval and oriented towards the mass spectrometry direction, and the spray solvent is evenly sprayed onto the surface of the brain section.

[0015] Preferably, for the method of detecting the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging, the brain section is in the sagittal plane, and the ion channel with m / z 303.2330 is selected for imaging. The brain regions include one or more of the fornix, corpus callosum, olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum.

[0016] Preferably, for the method of detecting the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging, the brain section is in the coronal plane, and the ion channel with m / z 309.2794 is selected for imaging. The brain region includes the dentate gyrus in the subregion of the hippocampus.

[0017] Preferably, for the method of detecting the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging, the nucleic acid hydrolysis products are hydrolysis products of RNA and / or DNA, including free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides. The free modified nucleosides include one or more combinations of N6-methyladenosine, N6,O2'-dimethyladenosine, inosine, N4-acetylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, N2,N2-dimethylguanosine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, pseudouridine;

[0018] The free nucleosides include one or more combinations of adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine;

[0019] The free modified nucleotides include one or more combinations of N6-methyl-2'-deoxyadenosine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-methyl-2'-deoxycytidine triphosphate, N6-methyl-2'-deoxyadenosine triphosphate, 5-formyl-2'-deoxycytidine triphosphate, 2'-deoxyinosine triphosphate, N6-methyladenosine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, pseudouridine triphosphate;

[0020] The free nucleotides include one or more combinations of 2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, thymidine monophosphate, 2'-deoxycytidine triphosphate, thymidine triphosphate, 2'-deoxyuridine triphosphate, adenosine monophosphate, 2'-deoxyguanosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine triphosphate, guanosine triphosphate, and uridine triphosphate.

[0021] According to another aspect of the present invention, there is also provided a system for analyzing the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging data, which includes a brain mass spectrometry image acquisition module, a data extraction module, and a data analysis module;

[0022] The brain mass spectrometry image acquisition module is used to acquire multiple sets of brain sagittal or coronal mass spectrometry diagrams and submit them to the data extraction module;

[0023] The data extraction module, based on the obtained brain mass spectrometry diagrams, takes nucleic acid hydrolysis products as the target substances, extracts the ion intensities corresponding to the target substances in different brain regions according to the brain structure, and transmits the ion intensities corresponding to the extracted target substances and the extracted brain region information to the data analysis module; the nucleic acid hydrolysis products are hydrolysis products of RNA and / or DNA, including one or more of free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides;

[0024] The data analysis module, based on the received ion intensities of the target substances and the corresponding extracted brain regions, performs differential analysis on the content of each target substance in different brain regions with the control group one by one and outputs the analysis results.

[0025] Preferably, for the system for analyzing the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging data, the brain mass spectrometry image is obtained according to the method described in the present invention.

[0026] Preferably, for the system for analyzing the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging data, when the brain is in the sagittal plane, the ion channel of m / z 303.2330 is selected for imaging, and the brain regions include one or more of the fornix, corpus callosum, olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum;

[0027] When the brain is in the coronal plane, the ion channel of m / z 309.2794 is selected for imaging, and the brain region includes the dentate gyrus in the sub-region of the hippocampus.

[0028] Generally speaking, compared with the prior art by the above technical solutions conceived by the present invention, since the present invention improves the injection needle and the spraying system, the brain slice mass spectrometry imaging ion map obtained can clearly identify more different brain regions and can achieve the following beneficial effects:

[0029] The method for detecting the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging provided by the present invention uses a spray needle capillary with a diameter of 10-30 microns. By adjusting the angle between the spray needle and the sample slide to 55°-57° and the height to 35-36.5 mm, compared with the existing mass spectrometry imaging technology, the mass spectrometry map obtained by the present invention can clearly identify different brain region structures such as the fornix and corpus callosum on the sagittal plane and the dentate gyrus in the subregion of the hippocampus on the coronal plane, can accurately detect and analyze the spatial distribution of nucleic acid hydrolysis products in different brain regions, and by improving the spray solvent system and constructing a mass spectrometry imaging database of nucleic acid hydrolysis products, this method has higher sensitivity and accuracy for detecting nucleic acid hydrolysis products in the brain.

[0030] The system for analyzing the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging data provided by the present invention extracts the ion intensity of the target substance corresponding to different brain regions according to the mass-to-charge ratio of the monitored ions of nucleic acid hydrolysis products, obtains the spatial distribution of nucleic acid hydrolysis products in different brain regions of the brain, and by analyzing the differences in the content of each nucleic acid hydrolysis product in the distribution of different brain regions between the experimental group and the control group, it is beneficial to screen and evaluate the therapeutic effects of different drugs on nucleic acid modification-related diseases and promote the drug research and development of related diseases. In particular, it can accurately extract the ion intensity of the target substance corresponding to 14 brain regions including the olfactory bulb, pons and medulla oblongata, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, ventral striatum, fornix, and corpus callosum, which is beneficial to quickly detect the spatial distribution of multiple nucleic acid hydrolysis products in the brain. Description of the Drawings

[0031] Figure 1 is a schematic diagram of the experimental process in Example 1;

[0032] Figure 2 is a comparison of mass spectrometry imaging ion maps of the m / z 309.2794 ion channel in the negative ion mode obtained by spraying with spray needle capillaries of different diameters, Figure 2 where A is a 100-micron spray needle capillary and B is a 20-micron spray needle capillary;

[0033] Figure 3 is a comparison of mass spectrometry imaging ion maps of the m / z 309.2794 ion channel in the negative ion mode obtained with different heights and angles of the spray needle and the slide, Figure 3 where A is the angle between the spray needle and the slide is 60° and the height is 37 mm, and B is the angle between the spray needle and the slide is 57° and the height is 36.5 mm;

[0034] Figure 4 is a schematic diagram of the experimental process in Example 2;

[0035] Figure 5are the ion intensities corresponding to the analytes detected with different spray solvents in the positive ion mode;

[0036] Figure 6 are the ion intensities corresponding to the analytes detected with different spray solvents in the negative ion mode;

[0037] Figure 7 is the schematic diagram of the experimental procedure in Example 3;

[0038] Figure 8 is the schematic diagram of the mouse brain slice patch;

[0039] Figure 9 is the schematic diagram of the spatial quantitative analysis process of nucleic acid hydrolysis products, Figure 9 wherein A is the schematic diagram of the brain structure in the sagittal plane of the brain, B is the mass spectrometry imaging ion map of the m / z 303.2330 ion channel in the negative ion mode, and C are the 14 selected brain regions;

[0040] Figure 10 is the mass spectrometry diagram of the 14 selected brain regions. In Figure 10, A is the mass spectrometry diagram of the olfactory bulb, cerebral cortex, anterior olfactory nucleus, and corpus callosum brain regions, B is the mass spectrometry diagram of the caudate putamen, ventral striatum, fornix, and basal forebrain brain regions, C is the mass spectrometry diagram of the hippocampus, thalamus, hypothalamus, and midbrain brain regions, and D is the mass spectrometry diagram of the cerebellum, pons, and medulla brain regions;

[0041] Figure 11 is the box plot of the differential analysis of the analyte methyluridine in different brain regions. Detailed implementation manners

[0042] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] Based on mass spectrometry as a detector, the present invention adopts mass spectrometry imaging technology to determine the monitoring ions of nucleic acid hydrolysis products such as modified nucleosides, nucleosides, modified nucleotides, and nucleotides through experiments, and qualitatively analyze the target nucleic acid hydrolysis products in the brain based on the mass-to-charge ratios of the monitoring ions of each analyte. It is found in the experiments that when a spray needle capillary with a diameter of 10 - 30 microns is used to spray the brain slices, the obtained mass spectrometry imaging ion map can clearly show the outer contour of the brain slices and the general structures of different brain regions. In particular, when the angle between the spray needle and the sample slide is 55° - 57° and the height is 35 - 36.5 mm, different brain region structures such as the dentate gyrus in the hippocampus in the coronal plane and the corpus callosum and fornix in the sagittal plane can be clearly identified.

[0044] Furthermore, by comparing 8 spray solvent systems, it was found that the spray solvent had an impact on the sensitivity of the detection method. The results showed that using an acetonitrile-water mixed solution as the spray solvent gave better detection results. In particular, when the volume ratio of acetonitrile to water was 8:2, the ion intensities of most nucleic acid hydrolysis products such as modified nucleosides, nucleosides, modified nucleotides, and nucleotide standards were relatively high, and their detection sensitivity was higher than that of other spray solvent systems.

[0045] The present invention provides a method for detecting the spatial distribution of nucleic acid hydrolysis products in the brain based on mass spectrometry imaging, specifically including the following steps:

[0046] (1) Sample preparation: Attach frozen brain sections to be measured onto a positively charged glass slide to obtain a sample glass slide.

[0047] (2) Mass spectrometry imaging: Use a spray needle capillary with a diameter of 10 - 30 microns, adjust the angle between the spray needle and the sample glass slide to 55° - 57°, and the height to 35 - 36.5 mm. Uniformly spray the spray solvent in the spray needle onto the surface of the brain section to perform in-situ desorption and ionization of the metabolites in the brain section, and then transport them into the mass spectrometer for separation and detection according to the ion mass-to-charge ratio.

[0048] (3) Spatial quantitative analysis of nucleic acid hydrolysis products: Based on the mass spectrometry imaging data of the obtained brain sections, using nucleic acid hydrolysis products as the detection target substances, extract the mass spectrometry diagrams corresponding to different brain regions, extract the mass spectrometry data according to the monitored ion mass-to-charge ratio corresponding to the nucleic acid hydrolysis products, obtain the ion intensities of each nucleic acid hydrolysis product in different brain regions, and perform spatial quantitative analysis of the nucleic acid hydrolysis products; the nucleic acid hydrolysis products are hydrolysis products of RNA and / or DNA, including one or more combinations of free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides.

[0049] In the present invention, spraying is performed under the spraying conditions in step (2) to detect the brain section, and the obtained mass spectrometry imaging ion map can clearly identify different brain region structures such as the corpus callosum and fornix in the sagittal plane and the dentate gyrus in the subregion of the hippocampus in the coronal plane; specifically:

[0050] The glass slide to be measured is placed on an automatic moving stage, and with the assistance of a high-speed air flow, the spray solvent is sprayed onto the sample surface drop by drop in extremely small droplets to perform in-situ desorption and ionization of the metabolites on the brain section, scan the entire sample row by row, and perform ion imaging on the obtained mass spectrometry data to obtain a mass spectrometry imaging ion map.

[0051] In some embodiments, a spray needle capillary with a diameter of 20 microns is used, and the angle between the spray needle and the glass slide to be measured is adjusted to 57°, and the height is 36.5 mm; preferably, the spray flow rate is controlled so that the spray point is elliptical and faces the mass spectrometry direction, and the spray solvent is evenly sprayed onto the surface of the brain section. The desorption of ions at the current sampling point will not affect the next sampling point, which is beneficial to further improving the spatial resolution of mass spectrometry imaging.

[0052] Furthermore, by comparing different spray solvent systems, the present invention finds that the type of organic solvent in the spray solvent and its ratio to water will affect the in-situ desorption and ionization of modified nucleosides, nucleosides, modified nucleotides, and nucleotides, thereby affecting the ion intensity of the target analyte to be measured. The experimental results show that the spray solvent is an acetonitrile-water mixed solution, in which the volume ratio of acetonitrile:water is (5-8):(2-5), which helps to improve the sensitivity and coverage of mass spectrometry imaging for detecting nucleic acid hydrolysis products such as modified nucleosides, nucleosides, modified nucleotides, and nucleotides. In particular, when the volume ratio of acetonitrile:water is 8:2, the detection sensitivity is significantly higher than that of other spray solvents.

[0053] In some embodiments, the brain is sectioned along the sagittal plane, and the ion channel of m / z 303.2330 in the negative ion acquisition mode is selected. According to the sagittal brain structure, the mass spectrometry maps corresponding to different brain regions are extracted. The brain regions include the olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, ventral striatum, fornix, and corpus callosum.

[0054] In some embodiments, the brain is sectioned along the coronal plane, and the ion channel of m / z 309.2794 in the negative ion acquisition mode is selected. According to the coronal brain structure, the mass spectrometry maps corresponding to different brain regions are extracted. The brain regions include the dentate gyrus in the subregion of the hippocampus.

[0055] In some embodiments, the nucleic acid hydrolysis products are hydrolysis products of RNA and / or DNA, including free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides;

[0056] The free modified nucleosides include one or more combinations of methyladenosine, N6, O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2, N2-dimethylguanosine, methyluridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, and pseudouridine;

[0057] The free nucleosides include one or more combinations of adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, and thymidine;

[0058] The free modified nucleotides include one or a combination of more than one of methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-methyl-2'-deoxycytidine triphosphate, N6-methyl-2'-deoxyadenosine triphosphate, 5-formyl-2'-deoxycytidine triphosphate, 2'-deoxyinosine triphosphate, methylcytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, N6-methyladenosine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, pseudouridine triphosphate;

[0059] The free nucleotides include one or a combination of more than one of 2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, thymidine monophosphate, 2'-deoxycytidine triphosphate, thymidine triphosphate, 2'-deoxyuridine triphosphate, adenosine monophosphate, 2'-deoxyguanosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine triphosphate, guanosine triphosphate, uridine triphosphate.

[0060] In some embodiments, the preferred spray solvent system is an acetonitrile-water mixed solution, wherein the volume ratio of acetonitrile to water is (5-8):(2-5), more preferably the volume ratio of acetonitrile to water is 8:2. Among the conditions of the mass spectrometry imaging system, the nitrogen pressure is 0.63 - 0.67 MPa, the spray liquid flow rate is 5 μl / min. Under the condition that the spray solvent system is an acetonitrile-water mixed solution, positive and negative full scans are performed, the mass-to-charge ratio scanning range is 78 - 780, and mass spectrometry data is obtained;

[0061] In the mass spectrometry imaging data collected in the negative ion mode, the ion channel of m / z 303.2330 is selected, and according to the sagittal brain structure, the mass spectrometry maps corresponding to different brain regions are extracted, and the ion intensities of free modified nucleosides, free nucleosides, free modified nucleotides and free nucleotides in different brain regions are obtained, and thus the quantitative analysis of the spatial distribution of nucleic acid hydrolysis products in the brain can be realized.

[0062] In addition, the present invention also provides a system for analyzing the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging data, which includes a brain mass spectrometry image acquisition module, a data extraction module and a data analysis module;

[0063] The brain mass spectrometry image acquisition module, according to whether the brain slice is sagittal or coronal, selects the corresponding ion channel for imaging in the negative ion acquisition mode, obtains the mass spectrometry map, and submits it to the data extraction module; in some embodiments, the brain slice is sagittal, the ion channel of m / z 303.2330 in the negative ion acquisition mode is selected for imaging, and according to the sagittal brain structure, the negative ion mass spectrometry maps corresponding to different brain regions are extracted; in some embodiments, the brain slice is coronal, the ion channel of m / z 309.2794 in the negative ion acquisition mode is selected for imaging, and according to the coronal brain structure, the negative ion mass spectrometry maps corresponding to different brain regions are extracted.

[0064] The data extraction module extracts mass spectra of different brain regions according to the sagittal or coronal brain structure, extracts mass spectrometry data based on the monitored ion mass-to-charge ratios of nucleic acid hydrolysis products, obtains the ion intensities of their hydrolysis products in different brain regions, and transmits the ion intensities of the extracted target substances and the corresponding brain region information to the data analysis module; the nucleic acid hydrolysis products are RNA and / or DNA hydrolysis products, including one or more of free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides. Specifically:

[0065] It is used to extract the ion intensities of the corresponding target substances in each brain region with nucleic acid hydrolysis products as the target substances based on the obtained positive and negative ion mass spectra of different brain regions of the brain, and transmit the ion intensities of the extracted target substances and the corresponding brain region information to the data analysis module.

[0066] In some embodiments, negative ion mass spectra corresponding to different brain regions are extracted according to the sagittal brain structure; the brain regions include one or more of the fornix, corpus callosum, olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum;

[0067] In some embodiments, negative ion mass spectra corresponding to different brain regions are extracted according to the coronal brain structure; the brain regions include the dentate gyrus, a subregion in the hippocampus.

[0068] The data analysis module performs differential analysis on the content of each target substance in different brain regions with respect to the control group one by one based on the received target substance ion intensity data and the corresponding extracted brain regions, and outputs the analysis results, such as drawing a box plot and outputting the analysis results.

[0069] In some embodiments, the nucleic acid hydrolysis products are RNA and / or DNA hydrolysis products, including free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides;

[0070] The free modified nucleosides include one or more combinations of methyladenosine, N6, O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2, N2-dimethylguanosine, methyluridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, pseudouridine;

[0071] The free nucleosides include one or more combinations of adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine;

[0072] The free modified nucleotides include one or a combination of more than one of methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-methyl-2'-deoxycytidine triphosphate, N6-methyl-2'-deoxyadenosine triphosphate, 5-formyl-2'-deoxycytidine triphosphate, 2'-deoxyinosine triphosphate, methylcytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, N6-methyladenosine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, pseudouridine triphosphate;

[0073] The free nucleotides include one or a combination of more than one of 2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, thymidine monophosphate, 2'-deoxycytidine triphosphate, thymidine triphosphate, 2'-deoxyuridine triphosphate, adenosine monophosphate, 2'-deoxyguanosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine triphosphate, guanosine triphosphate, uridine triphosphate.

[0074] Furthermore, the mass spectrometry imaging data of the brain slices is obtained according to the mass spectrometry imaging detection method described in the present invention:

[0075] Attach the cryosection of the brain sample to be tested to a positively charged glass slide to obtain the glass slide to be tested;

[0076] Use a spray needle capillary with a diameter of 20 microns, adjust the angle between the spray needle and the glass slide to be tested to 57°, and the height to be 36.5 mm, so that the spray point is oval and faces the mass spectrometry direction. Uniformly spray the spray solvent in the spray needle onto the surface of the brain slice to perform in-situ desorption and ionization of the metabolites of the brain slice, and then transport them into the mass spectrometer for separation and detection according to the mass-to-charge ratio of the ions to obtain the mass spectrometry imaging data of the brain slice.

[0077] Among them, the mass-to-charge ratio of the monitoring ions of the target analyte to be tested is crucial for the accuracy of this mass spectrometry imaging method. The present invention has established a mass spectrometry imaging database through experiments to identify free modified nucleosides, free nucleosides, free modified nucleotides and free nucleotides in the mouse brain, which is conducive to mapping their spatial distribution and content. This analysis system can be used to screen and evaluate the therapeutic effects of different drugs on RNA and / or DNA modification and metabolic disorders, or for the diagnosis of diseases related to RNA and / or DNA modification, such as diseases related to RNA modification, such as Alzheimer's disease.

[0078] The following are examples:

[0079] In the present invention, C57BL / 6J wild-type mice and 5-FAD Alzheimer's disease mice are used as experimental subjects, which are from the experimental animal center of the Beijing Institute of Brain Science and Brain-Inspired Intelligence. The animal experiments in the following examples have all been approved by the Animal Protection and Welfare Committee.

[0080] Example 1: Comparing the Effects of Different Parameters on the Spatial Resolution of Mass Spectrometry Imaging

[0081] Mass spectrometry imaging provides untargeted analysis in tissue sections. Hundreds of ion distribution patterns can be obtained in a single mass spectrometry imaging experiment, which can be used to reveal the in-situ spatial distribution of endogenous metabolites. In this example, the effects of changes in parameters such as the capillary diameter inside the spray needle, the height and angle between the spray needle and the glass slide on the spatial resolution of mass spectrometry imaging were compared, and optimized spray needle conditions were screened. The experimental procedure is as Figure 1 shown, and the specific experiments are as follows:

[0082] (1) Obtaining a mouse brain: The mouse was anesthetized by intraperitoneal injection of 2.5% avertin at a dose of 0.35 g / kg. After anesthesia and sacrifice, the mouse brain, including the olfactory bulb to the medulla region, was taken, placed on ice, and washed with pre-cooled phosphate buffered saline. The residual phosphate buffered saline on the surface of the brain sample was carefully blotted dry with dust-free paper, and then the mouse brain was placed in a cell culture dish and stored in a -80°C refrigerator for later use.

[0083] (2) Preparing brain sections: The mouse brain obtained in (1) was taken out and placed in the chamber of a -22°C cryostat for 20 minutes, then fixed on the sample holder. Since the dentate gyrus of the hippocampal subregion is in the coronal plane, in this example, the sections were cut along the coronal plane direction, with each section having a thickness of 20 μm, and the sections were attached to positively charged glass slides.

[0084] (3) Mass spectrometry imaging: Experiments were carried out using an aerodynamic assisted ionization mass spectrometry imaging device and an Orbitrap Exploris 480 mass spectrometer. Specifically: The glass slide to be measured was placed on an automatic moving stage and moved row by row. With the assistance of a high-speed air flow, the spray solvent was sprayed onto the sample surface as extremely small droplets row by row to desorb and ionize the metabolites on the brain sections in-situ, and then transported into the mass spectrometer for separation and detection according to the ion mass-to-charge ratio; among them, spray needles with capillary diameters of 100 μm and 20 μm were used for spraying respectively, the angle between the spray needle and the sample glass slide was 55°, and the height between the spray needle and the sample glass slide was 35 mm.

[0085] Among the conditions of the mass spectrometry imaging system, the nitrogen pressure was 0.63 - 0.67 MPa, the spray liquid flow rate was 5 μl / min, the horizontal x-direction scanning rate was set to 0.2 mm / s, and the vertical spacing in the y-direction was set to 0.1 mm; the temperature of the ion transfer tube was 350°C, the resolution was 1.2 million, negative ion full scan was performed, and the mass-to-charge ratio scanning range was 78 - 780; the automatic gain control target was 1E7, and the maximum injection time was 100 ms.

[0086] (4) Mouse brain slice imaging: The data was converted to the CDF format using Xcalibur software and then processed using MassImager software to subtract the background with a mass tolerance of 10 ppm. In the mass spectrometry imaging data collected in the negative ion mode, the mass spectrometry imaging data of the m / z 309.2794 ion channel was selected.

[0087] The above ion channel corresponds to an endogenous metabolite with a characteristic distribution in the mouse brain. The spatial resolution of the mass spectrometry imaging method was examined through its mass spectrometry imaging ion map, and then the mass spectrometry imaging ion maps obtained under different parameter scans were compared to determine the optimal needle spraying conditions and improve the spatial resolution of this mass spectrometry imaging method. Among them, the mass spectrometry imaging ion map of the m / z 309.2794 ion channel in the negative ion mode is as Figure 2 shown.

[0088] Among them Figure 2 in which A is the needle capillary with a diameter of 100 μm, the mass spectrometry imaging ion map of the m / z 309.2794 ion channel in the negative ion mode, B is the needle capillary with a diameter of 20 μm, and the mass spectrometry imaging ion map of the m / z 309.2794 ion channel in the negative ion mode.

[0089] It can be seen from Figure 2 that the ion map obtained by the 20-μm capillary can clearly show the outer contour and general structure of the brain slice, and its spatial resolution is much higher than that of the 100-μm capillary. In addition, the ion maps obtained by the 10-μm and 30-μm capillaries can clearly show the outer contour and general structure of the brain slice.

[0090] Furthermore, by continuously adjusting the angle (50° - 60°) and height (35 - 37 mm) between the needle and the sample slide, the shape and direction of the spray point were finely adjusted. The spray point needs to be small and stable, but not blurred or moving, which is crucial for stable ion signals.

[0091] By comparing the mass spectrometry imaging ion maps of m / z 309.2794 obtained under different parameters (the height and angle between the needle and the slide), the angles of 55° - 57° and the height of 35 - 36.5 mm were selected, which can clearly identify the different brain region structures on the coronal plane; the optimal angle is 57° and the height is 36.5 mm, which can clearly identify the different brain region structures on the coronal plane, and even the structure of the dentate gyrus in the subregion of the hippocampus, as Figure 3 shown.

[0092] Figure 3 In which A is the mass spectrometry imaging ion map of the m / z 309.2794 ion channel obtained with a 20-μm diameter needle capillary, the angle between the needle and the slide is 60°, and the height is 37 mm, and the structure of the dentate gyrus in the subregion of the hippocampus on the coronal plane cannot be clearly identified.

[0093] Figure 3 In this case, B is the diameter of the needle capillary, which is 20 microns. The angle between the needle and the glass slide is 57°, and the height is 36.5 mm. The mass spectrometry imaging ion map of the m / z 309.2794 ion channel obtained can clearly identify the different brain region structures in the coronal plane, even the structure of the dentate gyrus in the sub-region of the hippocampus, which can improve the spatial resolution of mass spectrometry imaging. Moreover, it is also found that the spray marking is oval and oriented towards the mass spectrometry direction, and the desorption of ions at the current sampling point will not affect the next sampling point, which is conducive to further improving the spatial resolution of mass spectrometry imaging.

[0094] In summary, the optimized detection conditions determined in this embodiment are that the diameter of the needle capillary is 20 microns, the angle between the needle and the glass slide is 57°, and the height is 36.5 mm. Under these detection conditions, different brain region structures in the coronal plane, such as the structure of the dentate gyrus in the sub-region of the hippocampus, can be clearly distinguished.

[0095] Example 2: Influence of Different Spray Solvents on the Detection Sensitivity of Mass Spectrometry Imaging

[0096] Based on the optimized detection conditions (the diameter of the needle capillary is 20 microns, the angle between the needle and the glass slide is 57°, and the height is 36.5 mm) screened in Example 1, this embodiment compared the effects of 8 spray solvents on the results of detecting free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides in mass spectrometry imaging. Among them, the 8 spray solvents are methanol-water (MeOH:H 2 O = 5:5), methanol-isopropanol-water (MeOH:IPA:H 2 O = 4:4:2), methanol-isopropanol-water (MeOH:IPA:H 2 O = 6:2:2), methanol-water (MeOH:H 2 O = 8:2), acetonitrile-water (ACN:H 2 O = 5:5), acetonitrile-isopropanol-water (ACN:IPA:H 2 O = 4:4:2), acetonitrile-isopropanol-water (ACN:IPA:H 2 O = 6:2:2), acetonitrile-water (ACN:H 2 O = 8:2), all in volume ratios.

[0097] The experimental procedure is as Figure 4 shown, and the specific experiment is as follows:

[0098] (1) Obtain the mouse brain: The same as in Example 1.

[0099] (2) Preparation of the test sample containing modified nucleosides and nucleoside standards: Grind the entire above-mentioned mouse brain and add the same volume of water to the obtained brain homogenate. Then, add 1 μL of 400 μM modified nucleosides and nucleoside standards to the brain homogenate to make the final concentration of the sample 200 μM, and apply it on the positively charged glass slide of the PVC adhesive tape with 2×5 mm rectangular holes. The modified nucleosides and nucleoside standards include cytidine, guanosine, methylcytidine, inosine, adenosine, uridine, pseudouridine, methylguanosine, and methyladenosine. Transfer 2 μL of the mixture to different rectangular holes respectively, and use the blank brain matrix as a negative control and add it on the same glass slide. The glass slide is dried in vacuum for subsequent mass spectrometry imaging scanning.

[0100] (3) Mass spectrometry imaging: Conduct experiments using an aerodynamic assisted ionization mass spectrometry imaging device and an Orbitrap Exploris 480 mass spectrometer. Among the conditions of the mass spectrometry imaging system, the nitrogen pressure is 0.63 - 0.67 MPa, the spray liquid flow rate is 5 μL / min, and the spray solvent systems are the above 8 kinds respectively.

[0101] The angle between the spray needle and the glass slide is 57°, the height is 36.5 mm, the horizontal x - direction scanning rate is set to 0.2 mm / s, the vertical pitch in the y - direction is set to 0.1 mm. For the connected mass spectrometer, the duration of this detection method is equal to the moving time of the mass spectrometry imaging sample platform. The temperature of the ion transfer tube is 350 °C, and the resolution is 1.2 million. Conduct positive and negative full scans, and the mass - to - charge ratio scanning range is 78 - 780. The automatic gain control target is 1E7, and the maximum injection time is 100 ms to obtain mass spectrometry data;

[0102] (4) Extract the ion intensity of nucleic acid hydrolysis products: Use Xcalibur software to convert the data into CDF format, and then use MassImager software for processing, subtract the background, and set the mass tolerance to 10 ppm. By analyzing the mass spectrometry imaging data obtained from scanning in both positive and negative modes, extract the ion intensity of the target substances under 8 spray solvent systems. The nucleic acid hydrolysis products include free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides. The detection results of some intercepted nucleic acid hydrolysis products are as Figure 5 and Figure 6 shown.

[0103] Figure 5 are the ion intensities corresponding to the detected target substances under different spray solvents in the positive ion mode; Figure 6 are the ion intensities corresponding to the detected target substances under different spray solvents in the negative ion mode.

[0104] From Figure 5 and Figure 6It can be seen that the spray solvent system is an acetonitrile-water mixed solution, where the volume ratio of acetonitrile to water is (5-8):(2-5). The ionic strength corresponding to each target is relatively high. Especially in the positive and negative ion acquisition modes, especially when the volume ratio of acetonitrile to water is 8:2, the detected ionic strength is significantly higher than that of other spray solvents, and the detection sensitivity is the highest.

[0105] Example 3 Construction of a mass spectrometry imaging database

[0106] The experimental procedure is as Figure 7 shown, and the specific experiment is as follows:

[0107] (1) Obtain a mouse brain: same as Example 1.

[0108] (2) Preparation of samples to be tested: Grind the entire mouse brain described above and add the same volume of water to the obtained brain homogenate. Then, add 1 μL of 400 μM modified nucleosides, nucleosides, modified nucleotides, and nucleotide standards to the brain homogenate respectively to make the final concentration of the sample 200 μM, and smear it on a positively charged glass slide with a PVC sticker having a 2×5 mm rectangular hole.

[0109] The above standards include modified nucleosides (methyladenosine, N6,O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2,N2-dimethylguanosine, methyluridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, pseudouridine); nucleosides (adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine); modified nucleotides (methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-methyl-2'-deoxycytidine triphosphate, N6-methyl-2'-deoxyadenosine triphosphate, 5-formyl-2'-deoxycytidine triphosphate, 2'-deoxyinosine triphosphate, methylcytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, N6-methyladenosine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, pseudouridine triphosphate) and nucleotides (2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, thymidine monophosphate, 2'-deoxycytidine triphosphate, thymidine triphosphate, 2'-deoxyuridine triphosphate, adenosine monophosphate, 2'-deoxyguanosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine triphosphate, guanosine triphosphate, uridine triphosphate).

[0110] Transfer 2 μL of the mixture to different rectangular holes respectively, and add the blank brain matrix as a negative control on the same glass slide. The glass slide is dried in vacuo for subsequent mass spectrometry imaging scans.

[0111] (3) Mass spectrometry imaging: Experiments were carried out using an aerodynamic assisted ionization mass spectrometry imaging device and an Orbitrap Exploris 480 mass spectrometer. In the conditions of the mass spectrometry imaging system, the nitrogen pressure was 0.65 MPa, the spray liquid flow rate was 5 μl / min, and the spray solvent system was an acetonitrile-water mixed solution (ACN:H 2 O = 8:2, volume ratio).

[0112] The horizontal angle between the glass slide and the spray needle was 57°, the height was 36.5 mm, the horizontal x-direction scanning rate was set to 0.2 mm / s, and the vertical spacing in the y-direction was set to 0.1 mm; the temperature of the ion transfer tube was 350 °C, and the resolution was 1.2 million. Positive and negative full scans were performed, and the mass-to-charge ratio scanning range was 78 - 780. The automatic gain control target was 1E7, and the maximum injection time was 100 ms.

[0113] (4) Determination of the monitoring ions of target modified nucleosides, nucleosides, modified nucleotides and nucleotides: Use Xcalibur software to convert the data into CDF format, and then use MassImager software for processing, subtract the background, and set the mass tolerance to 10 ppm. By analyzing the mass spectrometry imaging data obtained by scanning in both positive and negative modes, the monitoring ions of target modified nucleosides, nucleosides, modified nucleotides and nucleotides were determined as follows:

[0114] Based on the molecular weights of target modified nucleosides, nucleosides, modified nucleotides and nucleotides, the m / z values of their 9 adduct ions were calculated. The adduct ions in the positive ion mode include [M+H] + , [M+Na] + , [M+K] + , [M+NH4] + ,

[0115] [M+H-H 2 O] + and [M] + , and the adduct ions in the negative ion mode include [M-H] - , [M+Cl] - and [M-H-H 2 O] - . Finally, the m / z value with the highest peak intensity among the 9 adduct ions was selected as the monitoring ion of the compound. The monitoring ions of different target modified nucleosides, nucleosides, modified nucleotides and nucleotides are shown in the following table.

[0116] Table 1 Monitoring ions corresponding to different specific nucleosides and nucleotides

[0117]

[0118]

[0119] An adduct ion is formed by the interaction of a precursor ion with one or more atoms or molecules, and the resulting ion contains all the atomic constituents of the precursor ion and additional atoms from the associated atoms or molecules. Other compounds present in the sample mixture, known as the matrix, can also form adduct ions with the precursor ion, and the resulting adduct ions without the analyte belong to the category of background ions.

[0120] Then, MassImager software is used to analyze the mass spectrometry imaging data obtained by scanning in both positive and negative modes, extract the mass spectrometry data of the sample region, obtain the peak intensity corresponding to the ion based on the calculated m / z value of the adduct ion, select the adduct ion with the highest ion intensity as the monitored ion, and construct a database.

[0121] Example 4 Detection of the Spatial Distribution of Nucleic Acid Hydrolysis Products in the Mouse Brain

[0122] Experimental animals: 5-FAD Alzheimer's disease mice and C57BL / 6J wild-type mice were used as experimental subjects, which were sourced from the experimental animal center of the Beijing Institute of Brain Science and Brain-Inspired Intelligence.

[0123] In this example, the brains of 6 mice were detected in total, with 3 in the experimental group and 3 in the control group. The experimental group consisted of Alzheimer's disease mice, and the control group consisted of wild-type mice; gene knockout mice can also be used in the experimental group. The specific experiment is as follows:

[0124] (1) Obtain the brains of each group of mice: Specifically, the same as in Example 1;

[0125] (2) Prepare brain sections: Take out the mouse brains obtained above, place them in the chamber of a -22°C cryostat for 20 minutes, cut the left and right brains with a small knife, fix them on the sample holder. This method can identify brain regions on both the sagittal and coronal planes. Since the sagittal plane can expose 14 brain regions at one time, which is more than the number of brain regions exposed by the coronal plane, in this example, sections were made along the sagittal plane direction, with each section having a thickness of 20 micrometers, and the sections were attached to positively charged glass slides.

[0126] The brain sections of each group of mice were attached to positively charged glass slides as shown in Figure 8 One glass slide was attached with 6 mouse brain sections, which were derived from 6 mice (1 - 3 were in the experimental group, 4 - 6 were in the control group). Two more glass slides were attached in the same order, marked and stored in an -80°C refrigerator.

[0127] (3) Mass spectrometry imaging: Before the mass spectrometry imaging experiment, take out the glass slides from the -80°C refrigerator and then dry them at room temperature for 20 minutes. The mass spectrometry imaging was the same as in Example 2 to obtain the mass spectrometry data.

[0128] (4) Spatial quantitative analysis of nucleic acid hydrolysis products: Use Xcalibur software to convert the data into CDF format, and then use MassImager software for processing. Subtract the background, and set the mass tolerance to 10 ppm; the nucleic acid hydrolysis products are hydrolysis products of RNA and / or DNA, including free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides;

[0129] According to the sagittal brain structure schematic diagram as Figure 9 shown in A, select the m / z 303.2330 ion channel in the mass spectrometry imaging data collected in the negative ion mode as Figure 9 shown in B; use MassImager software to select 14 brain regions as Figure 9 shown in C. The brain regions specifically include the olfactory bulb, pons and medulla oblongata, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, ventral striatum, fornix, and corpus callosum; then use MassImager software to extract the mass spectrometry data of these 14 brain regions, as shown in Figure 10 (A - D); among them, A in Figure 10 is the mass spectrometry diagram of the olfactory bulb, cerebral cortex, anterior olfactory nucleus, and corpus callosum brain regions, B is the mass spectrometry diagram of the caudate putamen, ventral striatum, fornix, and basal forebrain brain regions, C is the mass spectrometry diagram of the hippocampus, thalamus, hypothalamus, and midbrain brain regions, and D is the mass spectrometry diagram of the cerebellum, pons and medulla oblongata brain regions.

[0130] To reduce the workload of data extraction, according to the written mass spectrometry imaging data analysis program, search for changed free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides in each brain region of the mouse brain. The mass spectrometry imaging data analysis program is as follows:

[0131] step 0: Automatically create an Excel file to store the mass spectrometry data of each brain region;

[0132] step 1: Select the ion intensity according to the monitored ions of the target substance;

[0133] step 2: Integrate the data of all Excel files;

[0134] step 3: Normalize the data of three technical replicates;

[0135] step 4: Compare the data of experimental group mice and control group mice, and perform differential analysis on the content of each target free modified nucleoside, free nucleoside, free modified nucleotide, and free nucleotide in these 14 brain regions one by one and draw box plots. The analysis result of the target substance methyluridine is as Figure 11 shown, and other target substances can be detected in these 14 brain regions and corresponding box plots can be drawn.

[0136] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting the spatial distribution of nucleic acid hydrolysis products in the brain based on mass spectrometry imaging, characterized in that, it includes the following steps: (1) Sample preparation: Attach the frozen section of the brain to be measured onto a positively charged glass slide to obtain a sample glass slide; (2) Mass spectrometry imaging: Use a spray needle capillary with a diameter of 10 - 30 microns, adjust the angle between the spray needle and the sample glass slide to 55° - 57°, and the height to 35 - 36.5 mm. Uniformly spray the spray solvent in the spray needle onto the surface of the brain section to perform in-situ desorption and ionization of metabolites on the brain section. According to whether the brain section is a sagittal plane or a coronal plane, select the corresponding ion channel for imaging in the negative ion acquisition mode to obtain mass spectrometry imaging data; (3) Spatial quantitative analysis of nucleic acid hydrolysis products: Based on the mass spectrometry imaging data obtained in step (2), extract the ion intensity of the target substance corresponding to different brain regions according to the monitored ion mass-to-charge ratio of the nucleic acid hydrolysis products to obtain the spatial distribution of the nucleic acid hydrolysis products in different brain regions of the brain; The nucleic acid hydrolysis products are hydrolysis products of RNA and / or DNA, including one or more of free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides.

2. The method for detecting the spatial distribution of nucleic acid hydrolysis products in the brain based on mass spectrometry imaging according to claim 1, characterized in that, the spray solvent is an acetonitrile - water mixed solution, where the volume ratio of acetonitrile: water is (5 - 8):(2 - 5); preferably, the volume ratio of acetonitrile: water is 8:

2.

3. The method for detecting the spatial distribution of nucleic acid hydrolysis products in the brain based on mass spectrometry imaging according to claim 2, characterized in that, use a spray needle capillary with a diameter of 20 microns, adjust the angle between the spray needle and the sample glass slide to 57°, and the height to 36.5 mm for spraying.

4. The method for detecting the spatial distribution of nucleic acid hydrolysis products in the brain based on mass spectrometry imaging according to claim 3, characterized in that, control the flow rate of the spray solvent so that the spray points are elliptical and face the mass spectrometry direction, and uniformly spray the spray solvent onto the surface of the brain section.

5. The method for detecting the spatial distribution of nucleic acid hydrolysis products in the brain based on mass spectrometry imaging according to any one of claims 1 to 4, characterized in that, the brain section is a sagittal plane, select the m / z 303.2330 ion channel for imaging, and the brain regions include one or more of the fornix, corpus callosum, olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum.

6. The method for detecting the spatial distribution of nucleic acid hydrolysis products in the brain based on mass spectrometry imaging according to any one of claims 1 to 4, characterized in that, the brain section is a coronal plane, select the m / z 309.2794 ion channel for imaging, and the brain region includes the dentate gyrus in the subregion of the hippocampus.

7. The method for detecting the spatial distribution of nucleic acid hydrolysis products in the brain based on mass spectrometry imaging according to claim 1, characterized in that, The nucleic acid hydrolysis products are hydrolysis products of RNA and / or DNA, including free modified nucleosides, free nucleosides, free modified nucleotides and free nucleotides, wherein the free modified nucleosides include one or more combinations of methyladenosine, N6, O2'-dimethyladenosine, inosine, methylcytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methylguanosine, N2, N2-dimethylguanosine, methyluridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, pseudouridine; The free nucleosides include one or more combinations of adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine; The free modified nucleotides include one or more combinations of methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-methyl-2'-deoxycytidine triphosphate, N6-methyl-2'-deoxyadenosine triphosphate, 5-formyl-2'-deoxycytidine triphosphate, 2'-deoxyinosine triphosphate, methylcytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, N6-methyladenosine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, pseudouridine triphosphate; The free nucleotides include one or more combinations of 2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, thymidine monophosphate, 2'-deoxycytidine triphosphate, thymidine triphosphate, 2'-deoxyuridine triphosphate, adenosine monophosphate, 2'-deoxyguanosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine triphosphate, guanosine triphosphate, uridine triphosphate.

8. A system for analyzing the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging data, characterized in that, it includes a brain mass spectrometry image acquisition module, a data extraction module and a data analysis module; The brain mass spectrometry image acquisition module is used to acquire multiple groups of brain sagittal or coronal mass spectrometry maps and submit them to the data extraction module; The data extraction module, based on the obtained brain mass spectrometry maps, takes nucleic acid hydrolysis products as target substances, extracts the ion intensities corresponding to the target substances in different brain regions according to the brain structure, and transmits the ion intensities corresponding to the extracted target substances and the extracted brain region information to the data analysis module; the nucleic acid hydrolysis products are hydrolysis products of RNA and / or DNA, including one or more of free modified nucleosides, free nucleosides, free modified nucleotides and free nucleotides; The data analysis module, based on the received ion intensities of the target substances and the corresponding extracted brain regions, performs differential analysis on the content of each target substance in different brain regions with the control group one by one and outputs the analysis results.

9. The system for analyzing the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging data according to claim 8, characterized in that, the brain mass spectrometry image is obtained by the method according to any one of claims 1 to 4.

10. The system for analyzing the spatial distribution of brain nucleic acid hydrolysis products based on mass spectrometry imaging data according to claim 9, characterized in that, The brain is in the sagittal plane, and the ion channel with m / z 303.2330 is selected for imaging. The brain regions include one or more of the fornix, corpus callosum, olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum; The brain is in the coronal plane, and the ion channel with m / z 309.2794 is selected for imaging. The brain region includes the dentate gyrus, a subregion of the hippocampus.

Images