Methods and systems for detecting nucleic acid metabolites

Abstract

The modification on the nucleic acid molecule is determined using a method that detects a free modified nucleic acid metabolite. The method is performed using a system.

Description

Technical Field

[0001] This disclosure belongs to the field of biotechnology. Specifically, this disclosure relates to a method and analytical system for detecting the spatial distribution of nucleic acid metabolites in the brain based on mass spectrometry imaging (MSI). This disclosure also relates to a method and system for detecting modifications on DNA and / or RNA molecules by detecting free nucleic acid metabolites. Background Technology

[0002] Numerous studies have demonstrated that RNA or DNA modifications are closely related to biological development and disease. For example, RNA modifications can regulate RNA translation and alter RNA stability. Posttranscriptional RNA modifications are dynamic and crucial for cellular function. Even slight abnormalities in their regulation can significantly impact RNA metabolism, which is essential for cellular function. Therefore, RNA modifications play a key role in various biological activities and mediate normal cellular function and development.

[0003] For example, some RNA modifications can improve mRNA stability and enhance its translation into proteins, while others can target mRNA for degradation and prevent its translation. RNA modifications are also involved in mRNA localization and alternative splicing. Among them, N6-methyladenosine (m... 6 A) is a modification widely present in mRNA. 6 The formation of A is catalyzed by a methyltransferase complex, with methyltransferase-like protein 3 (mettl3) as a key factor. Studies have shown that inactivation of mettl3 in the mouse nervous system leads to m 6 Deletion of A-modification, prolonged RNA half-life, and aberrant splicing events lead to dysregulation of gene expression across the entire transcriptome. In mettl3 conditional knockout mice, the cerebellum atrophies abnormally and fails to develop into cerebellar structures, resulting in balance disorders. Therefore, mettl3-mediated m... 6 A modification plays an important role in the development of the mammalian cerebellum.

[0004] Recent research suggests that key factors in Alzheimer's disease (AD) may be related to alterations in RNA modification. For example, in neurons of AD patients, as misfolded tau protein accumulates, mRNA on the RNA undergoes changes. 6 The level of modification A increased more than fourfold. Subsequently, marked with m... 6 A-modified RNA molecules specifically bind to misfolded tau proteins with the assistance of the RNA-binding protein HNRNPA2B1. This process may be associated with tau protein deposition in Alzheimer's disease. Simultaneously, during stress, m 6A-type modification can induce phase separation in cells, isolating some mRNAs involved in cell repair and thus inhibiting the synthesis of repair proteins, thereby preventing their participation in cell repair. In Alzheimer's disease, the stress granules generated by phase separation are permanent or persistent, so that under this pathological state, mRNAs encoding cell repair proteins are continuously suppressed, ultimately leading to abnormal cell metabolism and death. However, mRNA modification, except for mRNA... 6 A may include other modifiers besides m. It is currently unclear whether m is also included. 6 Do other RNA modifications besides A participate in AD progression, to what extent, and what is the spatial distribution of RNA modifications in different brain regions?

[0005] Nucleic acid metabolites include free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides. The spatial distribution of metabolites in the brain can reflect the metabolic and modification levels of nucleic acids in brain regions before degradation. However, existing fluorescence imaging or immunoimaging methods using antibodies to bind modified nucleosides are prone to false positive results. There are methods using liquid chromatography-tandem mass spectrometry to detect free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides in the mouse brain. However, in these methods, the detection is performed after homogenizing the entire brain, which is not only labor-intensive but also destroys the spatial distribution information of nucleic acid metabolites in the brain. Therefore, these methods are difficult to use for detecting the distribution of free modified (deoxy)nucleosides, free nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides in different brain regions. Furthermore, dissecting and separating different brain regions before homogenizing for detection is time-consuming and labor-intensive. The corpus callosum and fornix contain complex commissural fibers that are difficult to separate, making it even more challenging to locate brain subregions. The spatial distribution of nucleic acid metabolites in the brain plays a crucial role in the study of brain development and brain diseases. However, there are currently no reported methods for detecting the spatial distribution of free modified nucleosides and free modified (deoxy)nucleotides in the brain.

[0006] With the development of MSI technology, it can be combined with professional image processing software to directly analyze biological tissue sections, generating two-dimensional ion intensity maps of any compound with a specified mass-to-charge ratio (m / z). This enables high-throughput, comprehensive, and rapid analysis of the composition, relative abundance, and distribution of compounds in tissues. By obtaining the spatial distribution of biomarkers, potential biomarkers can be discovered and monitored. However, there are thousands of metabolites in the brain. In the brain metabolome, complex lipids account for the largest proportion, while (deoxy)nucleosides, (deoxy)nucleotides, and their analogues account for only about 2.4%. Furthermore, the properties of many endogenous metabolites are very similar to those of target nucleosides and (deoxy)nucleotides. Therefore, existing MSI technology struggles to clearly identify brain subregion structures and facilitates subsequent data analysis due to its limitations in recognizing low levels of free modified (deoxy)nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides. This hinders the accurate and rapid identification of multiple free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides in different brain regions. Therefore, researching ways to improve the spatial resolution of existing MSI technology for different brain regions will help to accurately identify more brain regions and rapidly detect the spatial distribution of nucleic acid metabolites in the brain. This will provide new research directions for studying the molecular, epigenetic, and epitranscriptional modification mechanisms of normal tissues or cells or certain diseases.

[0007] Current methods for detecting DNA and RNA modifications are laborious and cannot obtain comprehensive spatial information on the dynamic changes of different DNA and RNA modifications under different conditions. Furthermore, these methods miss other potentially critical modifications or more important regions. Summary of the Invention

[0008] To address the aforementioned deficiencies or improvement needs of existing technologies, this disclosure provides a method and system based on mass spectrometry imaging (MSI) for detecting DNA and / or RNA modifications by detecting free DNA and RNA metabolites. The method and system of this disclosure facilitate the spatial detection of unbound, free modified nucleic acid metabolites, revealing dynamic regional changes in DNA and RNA modifications.

[0009] According to one aspect, this disclosure provides the application of the detection of free modified nucleic acid metabolites in determining nucleic acid molecular modifications.

[0010] In some embodiments, this disclosure can be used (e.g., in situ) to detect free modified nucleic acid metabolites, preferably using mass spectrometry imaging (MSI).

[0011] In some embodiments, this disclosure can determine changes in modifications on nucleic acid molecules by detecting changes in free modified nucleic acid metabolites.

[0012] In some implementations, the modification may include any chemical modification that affects DNA and / or RNA metabolites, such as methylation, deoxygenation, acylation, etc.

[0013] According to another aspect, this disclosure provides a method for detecting modifications on nucleic acid molecules, which includes the step of identifying free modified nucleic acid metabolites.

[0014] In some implementations, the step of identifying free modified nucleic acid metabolites may include mass spectrometry imaging (MSI) of the sample to obtain MSI data of the free modified nucleic acid metabolites.

[0015] In some embodiments, nucleic acid metabolites may include any metabolites of RNA and / or DNA, and include one or more selected from free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides.

[0016] In some implementations, MSI may be performed using positive and / or negative mode scanning.

[0017] In some embodiments, MSI data may include the ionic strength and / or mass-to-charge ratio (m / z) of characteristic adducts of free nucleic acid metabolites in the sample. In some embodiments, the sample may be in the form of cell / tissue homogenate, cells, tissue sections, etc. In some specific embodiments, the sample may be spread on a glass slide.

[0018] In some embodiments, the method may further include a step of comparing the obtained MSI data with a control sample. In some embodiments, the control sample may be derived from a healthy subject. By comparing the obtained MSI data of the test sample with the control sample, it is possible to determine, for example, whether the distribution or amount of modified free nucleic acid metabolites has changed, which may reflect changes in DNA / RNA molecules.

[0019] In some embodiments, the method includes the following steps: 1) performing mass spectrometry imaging (MSI) on the sample to obtain MSI data under positive and / or negative mode scans; 2) extracting the MSI data to obtain the ionic intensity and / or mass-to-charge ratio (m / z) values ​​of characteristic adduct ions of DNA or RNA metabolites in the sample; and optionally 3) comparing the extracted ionic intensities with a control, or obtaining an MSI image.

[0020] In some embodiments, the ionic strength and / or m / z value of the characteristic adduct of a free nucleic acid metabolite can be determined using a standard containing that metabolite. In some embodiments, the standard may contain the metabolite in a matrix derived from the same source as the sample to be tested. For illustrative purposes only, if the sample to be tested is a brain sample (e.g., a brain slice), the matrix may be a brain homogenate or a brain slice; if the sample to be tested is cells, the matrix may be a cell suspension or cells.

[0021] In some embodiments, the characteristic adduct ion and / or m / z value of a free nucleic acid metabolite can be determined by a method comprising the following steps: a) performing mass spectrometry imaging (MSI) on a standard to obtain MSI data under positive and / or negative mode scans; b) extracting the MSI data; and c) selecting the adduct ion and / or m / z value with the highest peak intensity as the characteristic ion intensity and / or m / z value of the free nucleic acid metabolite.

[0022] In some embodiments, the adduct ion may include, but is not limited to, those selected from [M+H]. + [M+Na] + [M+K] + [M+NH4] + [M+H-H2O] + [M] + [MH] - [M+Cl] - and [MH-H2O] - One or more of them.

[0023] In some implementations, the adduct ions generated during positive mode scanning may include those selected from [M+H]. + [M+Na] + [M+K] + [M+NH4] + [M+H-H2O] + and [M] + One or more of them.

[0024] In some implementations, the adduct ions generated during negative mode scanning may include those selected from [MH]. - [M+Cl] - and [MH-H2O] - One or more of them.

[0025] In some embodiments, the m / z value range for positive and / or negative mode scanning can be from 0 to 1000. In some embodiments, the m / z value range for positive and / or negative mode scanning can be from 10 to 1000, preferably from 20 to 900, and more preferably from 30 to 800.

[0026] In a specific implementation, the m / z value of the characteristic adduct ion is selected from the values ​​listed in Table 1.

[0027] In some implementations, MSI may include the step of applying a spray solution to the sample.

[0028] In some embodiments, the spray liquid may comprise an acetonitrile-water mixture, wherein the volume ratio of acetonitrile to water is (1:10) to (10:1) or any value between therewith. In some embodiments, the spray liquid may comprise an acetonitrile-water mixture, wherein the volume ratio of acetonitrile to water is 1:10, 1:9.5, 1:9, 1:8.5, 1:8, 1:7.5, 1:7, 1:6.5, 1:6, 1:5.5, 1:5, 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1.5, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 9:1, or 10:1.

[0029] In some embodiments, a nozzle with a diameter of 0.1-100 μm or any value between therewith can be used to spray the aerosol, preferably 1-90 μm or any value between therewith, and more preferably 10-30 μm or any value between therewith. In some embodiments, a nozzle with a diameter of 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm or 100 μm can be used to spray the aerosol.

[0030] In some embodiments, the above method may further include the step of forming an MSI image through an ion channel having a certain m / z value, for example, any value in the range of 10 to 1000 or between, preferably any value in the range of 20 to 900 or between, more preferably any m / z value in the range of 30 to 800 or between.

[0031] In some embodiments, free nucleic acid metabolites may include one or more selected from free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides.

[0032] In some embodiments, free nucleic acid metabolites include 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, uridine, pseudouridine, adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine, methylcytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine triphosphate, 5-methyl-2'-deoxycytidine triphosphate, N6-methyl-2'- The free nucleic acid metabolites are selected from one or more of the following: deoxyadenosine triphosphate, 5-formyl-2'-deoxycytidine triphosphate, 2'-deoxyinosine triphosphate, methylcytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, N6-methyladenosine monophosphate, uridine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, uridine triphosphate, pseudouridine triphosphate, 2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, thymidine monophosphate, 2'-deoxyuridine triphosphate, adenosine monophosphate, 2'-deoxyguanosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine triphosphate, guanosine triphosphate, and uridine triphosphate. In some specific embodiments, these free nucleic acid metabolites may include the characteristic adduct ions and / or their m / z values ​​shown in Table 1.

[0033] In some embodiments, the method of this disclosure can detect one or more free nucleic acid metabolites at a time.

[0034] In some embodiments, the MSI may be an ionizing MSI. In some specific embodiments, the MSI may be a desorption electrospray ionization (DESI) MSI.

[0035] In some embodiments, the sample may be a biological sample. In some embodiments, the sample may be animal or human tissue, body fluid (e.g., saliva, blood, urine, lymph, etc.) or cells, and may be healthy or diseased. Examples of tissue samples may include, but are not limited to, testicular, prostate, liver, breast, colon, kidney, lymphatic, and brain tissue.

[0036] In some embodiments, the methods of this disclosure can perform in-situ and / or spatial detection of free modified nucleic acid metabolites in a sample. In some embodiments, the methods of this disclosure can detect changes in the modification of free nucleic acid metabolites, which can reflect, for example, changes in the modification of DNA or RNA molecules in different regions of a sample.

[0037] Those skilled in the art will understand that there are no particular limitations on the control and it can be selected according to the actual situation. In some embodiments, the control may be an untreated sample, while the sample may be processed. In some embodiments, the control may be derived from healthy subjects, while the sample may be derived from diseased subjects.

[0038] In some implementations, the spray flow rate is controlled so that the spray spot on the sample is elliptical.

[0039] According to another aspect, this disclosure provides a system for implementing the methods of this disclosure.

[0040] In some embodiments, the system may include: a mass spectrometry imaging (MSI) unit for performing mass spectrometry imaging (MSI) on a sample to obtain MSI data under positive and / or negative mode scans; an MSI data extraction unit for extracting MSI data to obtain the ionic intensity and / or mass-to-charge ratio (m / z) values ​​of characteristic adduct ions of DNA or RNA metabolites in the sample; and optionally, a data analysis unit for comparing the extracted ionic intensities with a control or obtaining an MSI image.

[0041] In some implementations, the system may perform the methods described above.

[0042] To achieve the above objectives, in some specific embodiments, this disclosure provides a method based on mass spectrometry imaging (MSI) for detecting the spatial distribution of nucleic acid metabolites in the brain, wherein the method includes the following steps: (1) Sample preparation: The frozen brain slices to be tested are mounted on a positively charged glass slide to obtain the sample glass slide; (2) MSI: The spray in the nozzle is evenly sprayed onto the surface of the brain slice using a nozzle capillary with a diameter of 10-30 micrometers; the angle between the nozzle and the sample slide is adjusted to 55°-57° and the height between them is 35-36.5 mm; the metabolites on the brain slice are desorbed and ionized in situ; and depending on whether the brain slice is sagittal or coronal, the corresponding ion channel is selected for imaging in negative ion acquisition mode to obtain MSI data; (3) Spatial quantitative analysis of nucleic acid metabolites: Based on the MSI data obtained in step (2), the ion intensity corresponding to the target in different brain regions is extracted according to the mass-to-charge ratio of nucleic acid metabolite ions, thereby obtaining the spatial distribution of nucleic acid metabolites in different brain regions. The nucleic acid metabolites include metabolites of RNA and / or DNA, and include one or more selected from free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy) nucleotides and free (deoxy) nucleotides.

[0043] Preferably, in the method for detecting the spatial distribution of brain nucleic acid metabolites based on MSI, the spray contains an acetonitrile-water mixture with a volume ratio of acetonitrile to water of (5~8):(2~5), preferably 8:2.

[0044] Preferably, in the method for detecting the spatial distribution of brain nucleic acid metabolites based on MSI, the spraying step includes using a 20-micrometer diameter spray needle capillary to adjust the angle between the spray needle and the sample slide to 57° and the height between them to 36.5 mm.

[0045] Preferably, in the method for detecting the spatial distribution of brain nucleic acid metabolites based on MSI, the spray flow rate is controlled so that the spray point is elliptical and the spray is uniformly sprayed onto the surface of the brain slice along the mass spectrometry direction.

[0046] Preferably, in the method for detecting the spatial distribution of brain nucleic acid metabolites based on MSI, the brain slices include a sagittal plane and are imaged using an m / z 303.2330 ion channel; and the brain slices include one or more brain regions selected from the fornix, corpus callosum, olfactory bulb, pons and medulla oblongata, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum.

[0047] Preferably, in the method for detecting the spatial distribution of brain nucleic acid metabolites based on MSI, the brain slices include the coronal plane and the m / z 309.2794 ion channel is selected for imaging; and the brain slices include the dentate gyrus of the hippocampus subregion.

[0048] Preferably, in the MSI-based method for detecting the spatial distribution of brain nucleic acid metabolites, the nucleic acid metabolites include RNA and / or DNA metabolites, and include free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides. The free modified (deoxy)nucleosides may include one or more selected from the group consisting 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.

[0049] Free (deoxy)nucleosides may include one or more selected from the following groups: adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, and thymidine.

[0050] Free modified (deoxy) nucleotides may include one or more selected from the group consisting 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, and pseudouridine triphosphate.

[0051] Free (deoxy)nucleotides may include one or more selected from the group consisting 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.

[0052] According to another aspect, this disclosure further provides a system based on MSI data for analyzing the spatial distribution of brain nucleic acid metabolites, the system comprising: The brain mass spectrometry image acquisition module is used to acquire multiple sets of mass spectrometry images of the brain in the sagittal or coronal plane and submit them to the data extraction module; The data extraction module is used to extract the ion intensity corresponding to target substances that are nucleic acid metabolites in different brain regions based on the obtained brain mass spectrometry images and the brain structure, and to transmit the ion intensity and extracted brain region information to the data analysis module; wherein, nucleic acid metabolites include RNA and / or DNA metabolites, and include one or more selected from free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides; and The data analysis module is used to perform differential analysis of target substance content between different brain regions and controls based on the ionic strength and brain region information corresponding to the extracted target substance, and output the analysis results.

[0053] Preferably, in a system based on MSI data for analyzing the spatial distribution of brain nucleic acid metabolites, the brain mass spectrometry image is obtained according to the method described in this disclosure.

[0054] Preferably, in a system based on MSI data for analyzing the spatial distribution of brain nucleic acid metabolites, the brain may include a sagittal plane and be imaged using an m / z 303.2330 ion channel; and the brain may include one or more brain regions selected from the fornix, corpus callosum, olfactory bulb, pons and medulla oblongata, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, and ventral striatum.

[0055] The brain may include the coronal plane, with the m / z 309.2794 ion channel selected for imaging; and the brain may include the dentate gyrus of the hippocampus subregion.

[0056] Overall, compared to existing technologies, the technical solution disclosed herein, through improvements to the nozzle and spray, can obtain MSI ion maps of brain slices that clearly identify more different brain regions. Furthermore, this disclosure has the following beneficial effects: The MSI-based method for detecting the spatial distribution of brain nucleic acid metabolites (also known as metabolites) disclosed in this disclosure uses a nozzle capillary with a diameter of 10-30 micrometers, and adjusts the angle between the nozzle and the sample slide to 55°-57°, with a height of 35-36.5 mm. Compared to existing MSI techniques, the mass spectrometry images obtained in this disclosure can clearly identify the structures of different brain regions, such as the fornix and corpus callosum in the sagittal plane, and the dentate gyrus in the hippocampus subregion in the coronal plane, and can therefore be used to accurately detect and analyze the spatial distribution of nucleic acid metabolites in different brain regions. Furthermore, the method of this disclosure, through improved spraying and the construction of an MSI database of nucleic acid metabolites, has higher sensitivity and accuracy for the detection of brain nucleic acid metabolites.

[0057] This disclosure provides a system based on MSI data for analyzing the spatial distribution of nucleic acid metabolites in the brain. Based on the monitored mass-to-charge ratio of nucleic acid metabolite ions, the system extracts the ion intensity corresponding to the target substance in different brain regions and obtains the spatial distribution of nucleic acid metabolites in different brain regions. This system can be used to screen and evaluate the therapeutic effects of different drugs on nucleic acid modification-related diseases by analyzing the differences in the content and distribution of multiple nucleic acid metabolites in different brain regions between experimental and control groups. Therefore, this disclosure can promote the research and development of drugs for related diseases. Specifically, this disclosure can accurately extract the ion intensity corresponding to the target substance in 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. It facilitates the rapid detection of the spatial distribution of multiple nucleic acid metabolites in the brain.

[0058] This disclosure provides high-resolution MSI ion maps for each sample. The methods and systems disclosed herein can be used to screen and evaluate the therapeutic effects of drugs, environmental conditions, and treatments on nucleic acid modification and diseases by analyzing differences in the content and distribution of multiple nucleic acid metabolites in different brain regions between experimental and control groups. Attached Figure Description

[0059] Figure 1 A schematic diagram of the experimental procedure for Example 1 is shown.

[0060] Figure 2A comparison of MSI ion maps obtained from the m / z 309.2794 ion channel using nozzle capillaries of different diameters is shown. Figure 2 A shows an MSI ion map obtained using a 100-micron nozzle capillary; Figure 2 B shows the MSI ion map obtained using a 20-micron nozzle capillary.

[0061] Figure 3 The comparison shows the MSI ion maps obtained from the m / z 309.2794 ion channel in negative ion mode at different heights and angles between the nozzle and the slide. Figure 3 A shows the MSI ion map obtained with a nozzle angle of 60° to the glass slide and a height of 37 mm; and Figure 3 B shows the MSI ion map obtained with an angle of 57° between the nozzle and the glass slide and a height of 36.5 mm.

[0062] Figure 4 A schematic diagram of the experimental procedure for Example 2 is shown.

[0063] Figure 5 The ion intensity of the target analytes detected by different sprays in positive ion mode is shown.

[0064] Figure 6 The display shows the ion intensity of the target substance detected by different spray liquids in negative ion mode.

[0065] Figure 7 A schematic diagram of the experimental process in Example 3 is shown.

[0066] Figure 8 A schematic diagram of the mounting of a mouse brain slice is shown.

[0067] Figure 9 A schematic diagram of the spatial quantitative analysis process of nucleic acid metabolites is shown. Figure 9 A shows a schematic diagram of the brain structure in the sagittal plane; Figure 9 B shows the MSI ion map obtained from the m / z 303.2330 ion channel in negative ion mode; Figure 9 C shows the 14 selected brain regions.

[0068] Figure 10 shows mass spectrometry images of 14 brain regions. Figure 10A Mass spectrometry images of brain regions including the olfactory bulb, cerebral cortex, anterior olfactory nucleus, and corpus callosum are displayed. Figure 10B Mass spectrometry images of brain regions including the caudate putamen, ventral striatum, fornix, and basal forebrain are displayed. Figure 10C Mass spectrometry images of brain regions including the hippocampus, thalamus, hypothalamus, and midbrain are displayed. Figure 10D Mass spectrometry images of brain regions including the cerebellum, pons, and medulla oblongata are displayed.

[0069] Figure 11 Box plot showing the results of differential analysis of the target substance methyluridine in different brain regions.

[0070] Figure 12 A box plot showing the results of differential analysis of nucleic acid metabolites determined by MSI according to one embodiment of the present disclosure.

[0071] Figure 13 The results of differential analysis of nucleic acid metabolites determined by LC-MS / MS according to one embodiment of the present disclosure are shown. Detailed Implementation

[0072] DNA, the fundamental biomolecule encoding genetic instructions, is composed of nucleic acids found in DNA and RNA. These nucleic acids undergo chemical modifications catalyzed by cell-specific enzymes, directly affecting genetic instructions and key cellular processes such as transcription and degradation. While research has primarily focused on DNA modifications affecting deoxycytidine (dC), exploration of other nucleic acid modifications is emerging. High-throughput sequencing methods, though valuable, can only indirectly infer modifications from sequencing patterns, making them prone to errors; in contrast, liquid chromatography-tandem mass spectrometry (LC-MS / MS) can directly identify and quantify modifications. LC-MS / MS allows for precise study of DNA and RNA modification changes after DNA and RNA are isolated, digested, and processed into individual nucleosides, and reliably quantifies and even identifies low-abundance and novel DNA and RNA modifications. However, the material requirements and labor-intensive nature of LC-MS / MS, including the need to process DNA and RNA into individual nucleosides, hinder its application in comprehensive spatial analysis of DNA and RNA modifications within tissues or organs.

[0073] Understanding the dynamic spatial variations of various modifications is crucial for revealing hotspots in functional epigenetics and epitranscriptomics, which cannot be achieved by focusing solely on specific modifications or regions, as this may miss key epigenetic or epitranscriptomical aspects. Therefore, this disclosure locates localized alterations in DNA and RNA modifications by identifying spatial variations in free modified nucleic acid metabolites. Since free modified nucleic acid metabolites present in cells originate from DNA and RNA degradation processes, localized changes in DNA and RNA modifications can reflect changes in DNA and RNA modifications within cells or tissues. Exploring free modified nucleic acid metabolites as indicators of localized modification alterations provides a promising screening method for identifying areas worthy of further investigation in epigenetic and epitranscriptomics research.

[0074] Currently, MSI technology, combined with specialized image processing software, can directly analyze biological tissue sections to generate two-dimensional ion intensity maps of compounds with specified mass-to-charge ratios (m / z), enabling high-throughput, comprehensive, and rapid analysis of the composition, relative abundance, and distribution of compounds in tissues. By acquiring the spatial distribution of biomarkers, potential biomarkers can be discovered and monitored.

[0075] In the prior art, MSI analysis of modified (deoxy)nucleotides / (deoxy)nucleotides has not been used to gain a deeper understanding of DNA and RNA modification changes. This application provides for the first time an application of using free modified (deoxy)nucleotides / (deoxy)nucleotides detected by MSI to determine modifications or modification changes in DNA and / or RNA molecules.

[0076] In some embodiments, to achieve spatial visualization of free nucleic acid metabolites, this disclosure develops a comprehensive mass spectrometry imaging (MSI) method based on desorption electrospray ionization (DESI), named DREAMS. In DESI, aqueous droplets, acting as a jet, are guided to move biological samples, continuously releasing molecules while mass spectrometry (MS) is ongoing, revealing the relative spatial distribution of biomolecules. DREAMS is optimized to specifically target the release, detection, and analysis of free, unmodified, and modified nucleic acid metabolites, while retaining the ability to detect a variety of other metabolites.

[0077] Furthermore, the methods and systems disclosed herein have become powerful tools for spatial detection of free modified nucleic acid metabolites, providing unprecedented perspectives on DNA and RNA modifications. These methods and systems offer valuable insights into epigenetic and transcriptomic regulation in both healthy and disease states, serving as key spatial screening tools for identifying target regions and potential hotspots of change in tissues, cells, etc. Therefore, for example, they can be readily applied to screen cells or tissues to determine the effects of drugs, treatments, or environmental stimuli on these cells or tissues, and to assess changes in their free and potential DNA and RNA modifications.

[0078] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure. Furthermore, the technical features involved in the following embodiments of this disclosure can be combined with each other as long as they do not conflict with each other.

[0079] This disclosure utilizes MSI technology with mass spectrometry as the detector to experimentally determine the monitoring ions of nucleic acid metabolites (e.g., modified (deoxy)nucleosides, modified (deoxy)nucleotides, and (deoxy)nucleotides), and qualitatively determines the nucleic acid metabolites of brain targets by detecting the mass-to-charge ratio of the monitoring ions of each target. Experiments revealed that in MSI ion maps obtained by spraying brain slices using a 10-30 micrometer diameter needle capillary, the outer contour of the brain slices and the approximate structure of different brain regions can be clearly seen. In particular, when the angle between the needle and the sample slide is 55°-57° and the height between them is 35mm-36.5mm, different brain structures, such as the dentate gyrus in the hippocampus subregion in the coronal plane and the corpus callosum and fornix in the sagittal plane, can be clearly identified.

[0080] In addition, eight sprays were compared. The results showed that the spray affects the sensitivity of the detection method. Acetonitrile-water mixture sprays, especially those with a volume ratio of acetonitrile to water of 8:2, showed better detection performance, producing higher ionic strengths for most nucleic acid metabolites (e.g., modified nucleosides, nucleosides, modified (deoxy)nucleotides, and (deoxy)nucleotide standards), thus exhibiting higher detection sensitivity compared to other sprays.

[0081] This disclosure provides an MSI-based method for detecting the spatial distribution of nucleic acid metabolites in the brain, specifically including the following steps: (1) Sample preparation: The frozen brain slices to be tested are attached to a glass slide, such as a positively charged glass slide, to obtain a sample glass slide; (2) MSI: The spray in the nozzle is uniformly sprayed onto the surface of the brain slice using a nozzle capillary with a diameter of 10-30 micrometers; the angle between the nozzle and the sample slide is adjusted to 55°-57° and the height between them is 35-36.5 mm; the metabolites on the brain slice are desorbed and ionized in situ; and the ions are transferred to the mass spectrometer for separation and detection according to the mass-charge ratio. (3) Spatial quantitative analysis of nucleic acid metabolites: Based on the obtained brain slice MSI data, mass spectrometry images corresponding to different brain regions were extracted with nucleic acid metabolites as the detection targets; mass spectrometry data were extracted based on the mass-to-charge ratio of the monitoring ions corresponding to the nucleic acid metabolites; the ionic intensities of each nucleic acid metabolite in different brain regions were obtained; and spatial quantitative analysis of nucleic acid metabolites was performed. Nucleic acid metabolites include metabolites of RNA and / or DNA, and include one or more selected from free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides.

[0082] In this disclosure, brain slices are examined by spraying using the spraying conditions defined in step (2). In the obtained MSI ion map, different brain regions, such as the corpus callosum and fornix in the sagittal plane and the dentate gyrus in the hippocampal subregion in the coronal plane, can be clearly identified.

[0083] Specifically, the glass slide to be tested is placed on an automated moving platform; a spray is applied to the sample surface in rows as tiny droplets with the assistance of a high-speed airflow; metabolites on the brain slices are desorbed and ionized in situ; the entire sample is scanned row by row; and the obtained mass spectrometry data is used for ion imaging to obtain an MSI ion map.

[0084] In some embodiments, a 20-micrometer diameter nozzle capillary is used, with the angle between the nozzle and the glass slide being tested adjusted to 57° and the height between them adjusted to 36.5 mm. Preferably, the spray flow rate is controlled so that the spray spot is elliptical and oriented towards the mass spectrometer. This allows the spray to be uniformly applied to the surface of the brain slice, and the ion desorption at the current sampling point does not affect the next sampling point, which is beneficial for further improving the spatial resolution of MSI.

[0085] Furthermore, this disclosure, through comparison of different sprays, found that the type of organic solvent in the spray and its ratio to water affect the in-situ desorption and ionization of modified (deoxy)nucleosides, modified (deoxy)nucleotides, and (deoxy)nucleotides, thereby affecting the ionic strength of the target analytes. Experimental results show that acetonitrile-water mixture sprays, with an acetonitrile:water volume ratio of (5~8):(2~5), help improve the sensitivity and coverage of MSI detection of nucleic acid metabolites such as modified (deoxy)nucleosides, modified (deoxy)nucleotides, and (deoxy)nucleotides. In particular, the spray with an acetonitrile:water volume ratio of 8:2 exhibits significantly higher detection sensitivity compared to other sprays.

[0086] In some implementations, the brain is sliced ​​along the sagittal plane. In negative ion acquisition mode, the m / z 303.2330 ion channel is selected, and mass spectrometry images corresponding to different brain regions are extracted based on the brain structure in the sagittal plane. These brain regions 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.

[0087] In some implementations, the brain is sliced ​​along the coronal plane. In negative ion acquisition mode, the m / z 309.2794 ion channel is selected, and mass spectrometry images corresponding to different brain regions, including the dentate gyrus in the hippocampus, are extracted based on the brain structure in the coronal plane.

[0088] In some embodiments, nucleic acid metabolites include RNA and / or DNA metabolites, including free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides.

[0089] Free modified nucleosides include one or more selected from the group consisting 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.

[0090] Free nucleosides include one or more selected from the following groups: adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, and thymidine.

[0091] Free modified (deoxy) nucleotides include one or more selected from the group consisting 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, and pseudouridine triphosphate.

[0092] Free (deoxy)nucleotides include one or more selected from the group consisting 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.

[0093] In some embodiments, the spray preferably contains an acetonitrile-water mixture with a volume ratio of acetonitrile:water of (5~8):(2~5), preferably 8:2. Positive and negative full scans are performed under the following conditions: MSI system set to nitrogen pressure of 0.63~0.67 MPa, spray flow rate of 5 μL / min, spray as an acetonitrile-water mixture, and scan mass-to-charge ratio range of 78~780, to obtain mass spectrometry data.

[0094] By selecting the m / z 303.2330 ion channel from MSI data collected in negative ion mode, mass spectrometry images corresponding to different brain regions are extracted based on the sagittal brain structure, and the ionic intensities of free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides in different brain regions are obtained, enabling quantitative analysis of the spatial distribution of brain nucleic acid metabolites.

[0095] Furthermore, this disclosure also provides a system based on MSI data for analyzing the spatial distribution of nucleic acid metabolites in the brain. The system includes a brain mass spectrometry image acquisition module, a data extraction module, and a data analysis module.

[0096] The brain mass spectrometry image acquisition module, depending on whether the brain slice includes a sagittal or coronal plane, selects the corresponding ion channel for imaging in negative ion acquisition mode to obtain a mass spectrometry image, which is then submitted to the data extraction module. In some embodiments, the brain slice includes a sagittal plane, and imaging is performed by selecting the m / z 303.2330 ion channel in negative ion acquisition mode. Based on the brain structure in the sagittal plane, negative ion mass spectrometry images corresponding to different brain regions are extracted. In some embodiments, the brain slice includes a coronal plane, and imaging is performed by selecting the m / z 309.2794 ion channel in negative ion acquisition mode. Based on the brain structure in the coronal plane, negative ion mass spectrometry images corresponding to different brain regions are extracted.

[0097] The data extraction module extracts mass spectrometry images of different brain regions based on sagittal or coronal brain structures, extracts mass spectrometry data based on the monitored ion mass-to-charge ratio of nucleic acid metabolites, obtains the ion intensity of metabolites in different brain regions, and transmits the extracted target ion intensity and the corresponding brain region information to the data analysis module. Nucleic acid metabolites include RNA and / or DNA metabolites, including one or more selected from free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides.

[0098] Specifically, the data extraction module is used to extract the ion intensity of the corresponding target ion in each brain region based on the obtained positive and negative ion mass spectrometry images of different brain regions and the mass-to-charge ratio of the target ion, with nucleic acid metabolites as the target ion; and transmits the extracted target ion intensity and the region information of the corresponding brain region to the data analysis module.

[0099] In some implementations, negative ion mass spectrometry images corresponding to different brain regions are extracted based on the brain structure in the sagittal plane, wherein the brain regions include one or more selected from the fornix, corpus callosum, olfactory bulb, pons and medulla oblongata, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain and ventral striatum.

[0100] In some implementations, negative ion mass spectrometry images corresponding to different brain regions are extracted based on the coronal brain structure, wherein the brain regions include the dentate gyrus of the hippocampus subregion.

[0101] The data analysis module is used to perform differential analysis of the content of each target substance in different brain regions and controls based on the received ion intensity data and the corresponding brain regions of the target substance, and output the analysis results, such as drawing box plots.

[0102] In some embodiments, nucleic acid metabolites include RNA and / or DNA metabolites, including free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides.

[0103] Free modified (deoxy)nucleosides include one or more selected from the group consisting 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.

[0104] Free (deoxy)nucleosides include one or more selected from the following groups: adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, and thymidine.

[0105] Free modified (deoxy) nucleotides include one or more selected from the group consisting 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, and pseudouridine triphosphate.

[0106] Free (deoxy)nucleotides include one or more selected from the group consisting 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.

[0107] Furthermore, the brain slice MSI data were obtained according to the MSI detection method of this disclosure: Frozen brain slices to be tested were mounted on positively charged slides to obtain the test sample slides; and A 20-micrometer diameter nozzle capillary was used to uniformly spray the spray onto the surface of the brain slices. The angle between the nozzle and the sample slide was adjusted to 57° and the height to 36.5 mm, and the spray spot was made elliptical and oriented towards the mass spectrometer. Metabolites on the brain slices were desorbed and ionized in situ. Based on the ion mass-to-charge ratio, the data were transferred to the mass spectrometer for separation and detection, thereby obtaining the MSI data of the brain slices.

[0108] Monitoring the mass-to-charge ratio of the target ion is crucial for the accuracy of this MSI method. This disclosure experimentally constructs an MSI database to identify free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy)nucleotides, and free (deoxy)nucleotides in the mouse brain. This facilitates mapping their spatial distribution and abundance. This analytical system can be used to screen and evaluate the therapeutic effects of different drugs or treatments on RNA and / or DNA modifications and metabolic disorders, screen the effects of different treatments on cells, homogenates, or tissues, or diagnose RNA and / or DNA modification-related diseases or their effects on cellular or tissue regions. Diseases related to RNA modification include, for example, Alzheimer's disease.

[0109] As is generally used in this document, the terms “basically composed of” and “composed of” are included in the meaning of the term “comprising”.

[0110] As is generally used herein, unless otherwise stated, the articles “a,” “an,” and “the” mean “at least one” or “one or more.”

[0111] As used herein, the terms "about" or "approximately" mean that, given the nature or precision of the measurement, the degree of error in a quantitative measurement is acceptable. Typical examples of such error may be within 20%, 10%, or 5% of a given value or range of given values. Alternatively, particularly in biological systems, the terms "approximately" and "approximately" refer to values ​​within an order of magnitude, which may be 5 times or 2 times the given value.

[0112] In the following description, certain details will be set forth to provide a better understanding of the various embodiments of this disclosure. However, those skilled in the art will understand that this disclosure can be practiced without these details. In other instances, well-known structures and methods associated with mass spectrometers and mass spectrometry methods may not be shown or described in detail to avoid unnecessarily obscuring the description of embodiments of this disclosure.

[0113] This disclosure describes various features, aspects, and advantages of various embodiments of a mass spectrometer, as well as methods of manufacturing and using the mass spectrometer. However, this disclosure includes many alternative embodiments that can be achieved by combining the various features, aspects, and advantages of the various embodiments described herein in any combination or sub-combination that may be useful to those skilled in the art.

[0114] Example For illustrative purposes only, the following examples were performed on brain slices from C57BL / 6J wild-type mice and 5-FAD Alzheimer's disease mice. The mice were obtained from the Laboratory Animal Center of the Chinese Institute of Brain Science (Beijing). The animal experiments in the following examples have been approved by the Animal Welfare and Protection Committee.

[0115] Example 1. Determination of spatial resolution parameters for mass spectrometry imaging (MSI) Mass spectrometry imaging provides non-targeted analysis in tissue sections or on differentiated cultured cells. A range of different ion distribution maps can be obtained in a single MSI experiment and can be used to reveal the in-situ spatial distribution of endogenous metabolites. This example compares the effects of variations in various parameters (such as the capillary diameter of the nozzle, the height and angle between the nozzle and the slide) on the spatial resolution of MSI to screen for optimized nozzle parameters. The experimental procedure is as follows: Figure 1 As shown. The specific experiment was conducted as follows:

[0116] (1) Obtaining mouse brains: Mice were anesthetized by intraperitoneal injection of 2.5% avidin at a dose of 0.35 g / kg; after sacrifice, the mouse brains, including the olfactory bulb to the medulla oblongata region, were 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 with lint-free paper; the mouse brains were placed in cell culture dishes and stored in a -80°C freezer for later use.

[0117] (2) Preparation of brain slices: Take out the mouse brain obtained in step (1); place it in the chamber of a -22℃ cryostat for 20 minutes; fix it on the sample holder; since the dentate gyrus in the hippocampus is located on the coronal plane, this embodiment slices along the coronal plane, and each slice is 20 micrometers thick; and attach the slices to a positively charged glass slide.

[0118] (3) MSI: Experiments were conducted using an aerodynamically assisted ionization MSI device and an Orbitrap Exploris 480 mass spectrometer. Specifically: the test slide was placed on an automatic moving stage and moved row by row; the spray solution was sprayed onto the sample surface in tiny droplets with the assistance of a high-speed airflow; metabolites on the brain slices were desorbed and ionized in situ; and then transported to the mass spectrometer for separation and detection based on the ion mass-to-charge ratio. The spray solution was applied under the following conditions: the capillary diameters of the spray needle were approximately 100 micrometers and 20 micrometers, respectively; the angle between the spray needle and the sample slide was 55°; and the height between the spray needle and the sample slide was 35 mm.

[0119] The MSI system was set to nitrogen pressure 0.63-0.67 MPa, spray flow rate 5 μl / min, horizontal x-axis scan rate 0.2 mm / s, y-axis vertical spacing 0.1 mm, ion transmission tube temperature 350℃, resolution 1.2 million, negative ion full scan, mass-to-charge ratio scan range 78 to 780; automatic gain control (AGC) target 1E7, maximum injection time 100 ms.

[0120] (4) Mouse brain slice imaging: The data were converted to CDF format using Xcalibur software; the data were processed using MassImager software to remove the background, and the quality tolerance was set to 10 ppm. MSI data of the m / z 309.2794 ion channel were selected from the MSI data acquired in negative ion mode.

[0121] The aforementioned ion channels correspond to endogenous metabolites with characteristic distributions in the mouse brain. The spatial resolution of the MSI method is tested using its MSI ion map. Then, MSI ion maps obtained by scanning with different parameters are compared to determine the optimal nozzle conditions, thereby improving the spatial resolution of the MSI method. The MSI ion map obtained from the m / z 309.2794 ion channel in negative ion mode is shown below. Figure 2 As shown.

[0122] Figure 2 A shows the MSI ion map obtained using a 100 μm diameter nozzle capillary in negative ion mode with an ion channel at m / z 309.2794; and Figure 2 B shows the MSI ion map obtained using a 20 μm diameter nozzle capillary in negative ion mode with an ion channel of m / z 309.2794.

[0123] Depend on Figure 2 It can be seen that the ion maps obtained using a capillary with a diameter of 20 μm can clearly show the outer contour and general structure of the brain slices, and its spatial resolution is much higher than that obtained using a capillary with a diameter of 100 μm. In addition, the ion maps obtained using capillary diameters of 10 μm and 30 μm can also clearly show the outer contour and general structure of the brain slices.

[0124] Furthermore, the shape and direction of the spray point are fine-tuned by continuously adjusting the angle (50° to 60°) and height (35 mm to 37 mm) between the nozzle and the sample slide. The spray point should be small and stable, but not blurred or shifting, which is crucial for obtaining a stable ion signal.

[0125] By comparing MSI ion maps with m / z 309.2794 obtained under different parameters (height and angle between the nozzle and the slide), it was determined that an angle of 55°–57° and a height of 35–36.5 mm can clearly identify different brain regions on the coronal plane. The optimal angle is 57° and the optimal height is 36.5 mm. Using these parameters, different brain regions on the coronal plane, even the structures of the dentate gyrus in the hippocampal subregion, can be clearly identified. Figure 3 As shown.

[0126] Figure 3 A shows an MSI ion map obtained from the m / z 309.2794 ion channel using a 20 μm diameter nozzle capillary with a 60° angle between the nozzle and the slide and a height of 37 mm. Under these conditions, the structure of the dentate gyrus in the coronal hemaya region cannot be clearly identified.

[0127] Figure 3 B shows the MSI ion map obtained from the m / z 309.2794 ion channel using a 20 μm diameter nozzle capillary, with the nozzle at a 57° angle to the slide and a height of 36.5 mm. Under these conditions, different brain regions on the coronal plane, even the structures of the dentate gyrus in the hippocampal subregion, can be clearly identified. Therefore, the spatial resolution of MSI can be improved. It was also found that the spray point is elliptical and oriented towards the mass spectrometer, meaning that the desorption of ions at the current sampling point does not affect the next sampling point, thus further improving the spatial resolution of MSI.

[0128] In summary, the optimal detection conditions determined in this embodiment include: a nozzle capillary diameter of 20 micrometers, an angle of 57° between the nozzle and the slide, and a height of 36.5 mm. Under these detection conditions, different brain regions on the coronal plane can be clearly distinguished, such as the structures of the dentate gyrus in the hippocampus subregion.

[0129] Example 2. Effect of different spray solutions on MSI detection sensitivity In this embodiment, based on the optimized detection conditions selected in Example 1 (needle capillary diameter of 20 micrometers, angle between the needle and the glass slide of 57°, and height between them of 36.5 mm), the effects of eight spray solutions on MSI detection of free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides were compared. The eight spray solutions were methanol-water (MeOH:H2O=5:5), methanol-isopropanol-water (MeOH:IPA:H2O=4:4:2), methanol-isopropanol-water (MeOH:IPA:H2O=6:2:2), methanol-water (MeOH:H2O=8:2), acetonitrile-water (ACN:H2O=5:5), acetonitrile-isopropanol-water (ACN:IPA:H2O=4:4:2), acetonitrile-isopropanol-water (ACN:IPA:H2O=6:2:2), and acetonitrile-water (ACN:H2O=8:2). All of the above are volume ratios.

[0130] The experimental procedure is as follows Figure 4 As shown. The specific experiment was conducted as follows:

[0131] (1) Obtaining mouse brains: Same as in Example 1.

[0132] (2) Preparation of test samples containing modified nucleosides and nucleoside standards: Grind the entire mouse brain as described above 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 sample concentration 200 μM; spread it on a positively charged glass slide with 2×5 mm rectangular wells. The modified nucleosides and nucleoside standards include cytidine, guanosine, methylcytidine, inosine, adenosine, uridine, pseudouridine, methylguanosine, and methyladenosine. 2 μL of the mixture is pipetted into each rectangular well, and a blank brain matrix is ​​added as a negative control to the same glass slide. The glass slide is vacuum dried for subsequent MSI scanning.

[0133] (3) MSI: Experiments were conducted using an aerodynamically assisted ionization MSI apparatus and an Orbitrap Exploris 480 mass spectrometer. For the MSI system, the nitrogen pressure was set to 0.63-0.67 MPa and the spray flow rate to 5 μl / min. Samples were sprayed with the eight spray solutions mentioned above.

[0134] The angle between the nozzle and the glass slide was 57°, and the height between them was 36.5 mm. The horizontal x-axis scan rate was set to 0.2 mm / s. The vertical spacing in the y-axis was set to 0.1 mm. For the connected mass spectrometer, the detection duration was equal to the MSI sample platform travel time. The ion transfer tube temperature was set to 350 °C, and the resolution was set to 1.2 million. Positive and negative full scans were performed, with a mass-to-charge ratio scan range of 78-780. The automatic gain control target was set to 1E7. The maximum injection time was set to 100 ms to obtain mass spectrometry data.

[0135] (4) Extraction of ionic intensity of nucleic acid metabolites: Data were converted to CDF format using Xcalibur software; processed using MassImager software to subtract background, with a quality tolerance of 10 ppm. Ionic intensity of target analytes was extracted by using eight different spray solutions and analyzing MSI data obtained from scanning in both positive and negative modes. Nucleic acid metabolites included free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides. A portion of the nucleic acid metabolite detection results were extracted and... Figure 5 and Figure 6 The species are shown.

[0136] Figure 5 The display shows the ion intensity of the target analytes detected by different sprays in positive ion mode. Figure 6 The display shows the ion intensity of the target substance detected by different spray liquids in negative ion mode.

[0137] Depend on Figure 5 and Figure 6 It can be seen that when the spray liquid is an acetonitrile-water mixture with a volume ratio of acetonitrile to water of (5~8):(2~5), the ionic strength of each target analyte is relatively high, especially in both positive and negative ion collection modes. When the volume ratio of acetonitrile to water is 8:2, the obtained ionic strength is significantly higher than that of other spray liquids, and therefore it has the highest detection sensitivity.

[0138] Example 3. Construction of the MSI Database The experimental procedure is as follows Figure 7 As shown. The specific experiment was conducted as follows:

[0139] (1) Obtaining mouse brains: Same as in Example 1.

[0140] (2) Preparation of test samples: Grind the whole mouse brain as described above; add the same volume of water to the obtained brain homogenate; add 1 μL of 400 μM modified nucleosides, nucleosides, modified nucleotides and nucleotide standards to the brain homogenate to make the final sample concentration 200 μM; and spread it on a positively charged glass slide with PVC adhesive paper with 2×5 mm rectangular holes.

[0141] The 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, and pseudouridine); nucleosides (adenosine, 2′-deoxyguanosine, cytidine, guanosine, uridine, 2′-deoxyadenosine, 2'-deoxycytidine, and 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 and 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 and uridine triphosphate).

[0142] 2 μL of the mixture was pipetted into each rectangular well. Blank brain matrix was added as a negative control onto the same slide. The slide was vacuum-dried for subsequent MSI scanning.

[0143] (3) MSI: Experiments were conducted using an aerodynamically assisted ionization MSI apparatus and an Orbitrap Exploris 480 mass spectrometer. For the MSI system, the nitrogen pressure was set to 0.65 MPa and the spray flow rate to 5 μl / min. An acetonitrile-water mixture (ACN:H2O = 8:2, volume ratio) was used as the spray solution.

[0144] The horizontal angle between the slide and the nozzle is 57°, and the height between them is 36.5 mm. The horizontal x-axis scan rate is set to 0.2 mm / s. The vertical spacing in the y-axis is set to 0.1 mm. The ion transmission tube temperature is 350℃, and the resolution is set to 1.2 million. Positive and negative full scans are performed, with a mass-to-charge ratio scan range of 78-780. The automatic gain control target is set to 1E7. The maximum injection time is set to 100 ms.

[0145] (4) Identification of target modified nucleosides, nucleosides, modified nucleotides, and nucleotide monitoring ions: The data was converted to CDF format using Xcalibur software; processed using MassImager software to subtract background, with a quality tolerance of 10 ppm; and the target modified nucleosides, nucleosides, modified nucleotides, and nucleotides were identified by analyzing the MSI data obtained from scanning in positive and negative modes, as detailed below: Based on the molecular weight screening of the target modified nucleosides, nucleosides, modified nucleotides, and nucleotides, the m / z values ​​of nine adduct ions for specific DNA / RNA metabolites were calculated. Adduct ions generated in positive ion mode include [M+H]. + [M+Na] + [M+K] + [M+NH4] + [M+H-H2O] + and [M] + The adduct ions generated in negative ion mode include [MH]. - [M+Cl] - and [MH-H2O] - Ultimately, the adduct ion with the highest m / z value among the nine adduct ions was selected as the monitoring ion for specific DNA / RNA metabolites. The monitoring ions for different target substances, namely modified nucleosides, nucleosides, modified nucleotides, and nucleotides, are shown in the table below.

[0146] Table 1. Monitoring ions corresponding to specific nucleosides and nucleotides

[0147] Adduct ions are formed by the interaction of precursor ions with one or more atoms or molecules. The resulting ion contains all the atoms of the precursor ion as well as additional atoms of the associated atoms or molecules. Other compounds present in the sample mixture, called the matrix, can also form adduct ions with the precursor ions. The resulting analyte-free adduct ions are considered background ions.

[0148] Then, the MSI data obtained from scanning in both positive and negative modes were analyzed using MassImager software. Mass spectrometry data from the sample region were extracted, and the peak intensity corresponding to each ion was obtained based on the calculated m / z value of the adduct ion. Furthermore, the adduct ion with the highest intensity was selected as the monitoring ion, and a database was established.

[0149] Example 4. Detection of the spatial distribution of nucleic acid metabolites in the mouse brain Experimental animals: 5-FAD Alzheimer's disease mice and C57BL / 6J wild-type mice were used as experimental subjects, which were obtained from the Experimental Animal Center of Beijing Institute of Brain Science and Brain Inspired Intelligence.

[0150] In this embodiment, the brains of six mice were examined, with three Alzheimer's disease mice serving as the experimental group and three wild-type mice as the control group. Gene knockout mice could also be used as the experimental group. The specific experiment was conducted as follows:

[0151] (1) Obtaining the brains of each group of mice: Same as in Example 1.

[0152] (2) Preparation of brain slices: Take out the mouse brain obtained above; place it in the chamber of a -22℃ cryostat for 20 minutes; cut the brain in half with a blade and fix it to the sample holder; in this embodiment, slice along the sagittal direction, and each slice is 20 micrometers thick (because this method can distinguish the sagittal and coronal brain regions at the same time, and the sagittal plane can expose 14 brain regions at one time, more than the brain regions exposed in the coronal plane); attach the slices to a positively charged glass slide.

[0153] like Figure 8 As shown, mouse brain slices from each group were mounted on positively charged glass slides. Six mouse brain slices, from six mice (slices 1-3 were the experimental group, and slices 4-6 were the control group), were mounted on each slide in the same order, labeled, and stored at -80°C.

[0154] (3) MSI: Before the MSI experiment, the slide was taken out of the -80℃ refrigerator; dried at room temperature for 20 minutes; and MSI was performed in the same manner as in Example 2 to obtain mass spectrometry data.

[0155] (4) Spatial quantitative analysis of nucleic acid metabolites: The data were converted to CDF format using Xcalibur software and then processed using MassImager software to subtract background. The quality tolerance was set to 10 ppm. Nucleic acid metabolites include metabolites of RNA and / or DNA, such as free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides.

[0156] A schematic diagram of the sagittal brain structure is shown below. Figure 9 As shown in Figure A. The image was obtained by selecting the m / z 303.2330 ion channel (corresponding to arachidonic acid) from the MSI data acquired in negative ion mode. Figure 9 As shown in B. Fourteen brain regions were selected using MassImager software, as shown... Figure 9As shown in Figure C, the brain regions 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. MSI data for these 14 brain regions were then extracted using MassImager software. Figure 10A -As shown in 10D, Figure 10A Mass spectrometry images of brain regions including the olfactory bulb, cerebral cortex, anterior olfactory nucleus, and corpus callosum are displayed. Figure 10B Mass spectrometry images of brain regions including the caudate putamen, ventral striatum, fornix, and basal forebrain are displayed. Figure 10C Mass spectrometry images of brain regions including the hippocampus, thalamus, hypothalamus, and midbrain were displayed; and Figure 10D Mass spectrometry images of brain regions including the cerebellum, pons, and medulla oblongata are displayed.

[0157] To reduce the workload of data extraction, based on the developed MSI data analysis program, altered free modified nucleosides, free nucleosides, free modified nucleotides, and free nucleotides were searched in various brain regions of mice. The MSI data analysis program is shown below: Step 0: Automatically create an Excel file to store mass spectrometry data for all target brain regions; Step 1: Select the ion intensity based on the monitored ions of the target analyte; Step 2: Integrate the data from all Excel files; Step 3: Normalize the data using at least three sets of techniques or biological replicates; Step 4: Compare the data from the experimental group and the control group mice, and perform differential analysis on the content of each target free modified nucleoside, free nucleoside, free modified nucleotide, or free nucleotide in these 14 brain regions to generate box plots. The analysis results for the target methyluridine are shown exemplarily in... Figure 11 In this study, other targets can be detected in these 14 brain regions and generate corresponding box maps.

[0158] Example 5. Changes in free modified nucleosides detected by MSI can reflect changes in RNA molecule modification. This embodiment used the brains of 6 mice, including 3 conditionally knocked-out Pus7 mice (KO) as the experimental group and 3 Pus7 loxP mice as the control group. The specific experiment was conducted as follows:

[0159] (1) Generation of Pus7 knockout mice and loxP mice: Adenoma-associated plasmids pAAV-EF1α-Cre-P2A-EGFP (AAV-Cre-EGFP) and pAAV-EF1α-EGFP (AAV-EGFP) were constructed. For AAV-Cre-EGFP, the CRE recombinase coding sequence (Uniprot: P06956) was cloned into the AAV vector (Addgene) carrying the human elongation factor-1α (EF-1α) promoter. Enhanced green fluorescent protein (EGFP, Uniprot: A0A6G6D467) was cloned into the C-terminus of Cre and co-expressed by linking it with the P2A peptide. For AAV-EGFP, the vector did not contain P2A or Cre. Adeno-associated virus (AAV) was prepared from these constructs: Each plasmid was co-transfected into HEK293T cells in the presence of polyethyleneimine with the rep / cap-containing plasmid pUCmini-iCAP-PHP.eB (Addgene #103005) and the helper plasmid pAdDeltaF6 (Addgene #112867). The generated AAV virus was harvested after 72 hours, purified in chloroform, and titrated and quantified by qPCR. Mice were anesthetized with 0.35 g / kg of 2.5% avidin via intraperitoneal injection before infection. 100 μL of AAV-Cre-EGFP virus was injected intravenously via the retroorbital vein to obtain the brains of Pus7 knockout mice. 100 μL of AAV-EGFP virus was used as a control. Three biological replicates (n=3) were set up for each virus.

[0160] (2) Obtaining mouse brains: Same as in Example 1.

[0161] (3) Preparation of brain slices: Same as in Example 4.

[0162] (4) MSI: Same as Example 4.

[0163] (5) Spatial quantification of nucleic acid metabolites: Same as in Example 4. Results are as follows: Figure 12 As shown.

[0164] (6) LC-MS / MS quantification of pseudouridine RNA modification: Total RNA was extracted from mouse brains using Trizol (Thermo Fisher) reagent. 1 μg of RNA was added to a 20 μL reaction volume and digested at 37°C for 1 hour using a nucleoside digestion mixture (New England Biolabs, M0649S). The obtained nucleosides were analyzed by LC-MS / MS using a Vanquish Flex UHPLC system (Thermo Fisher) coupled with a TSQAltis™ triple quadrupole mass spectrometer (Thermo Fisher). Analysis of pseudouridine and overall uridine levels was performed on a TSQ Altis™ triple quadrupole mass spectrometer (Thermo Fisher) coupled with a Vanquish Flex UHPLC system (Thermo Fisher) equipped with an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm particle size, Waters). The mobile phase consisted of a 0.5% formic acid aqueous solution (solvent A) and a 0.5% formic acid acetonitrile solution (solvent B), with a flow rate of 300 μL / min. Calibration curves were generated using serial dilutions of synthetic standards of uridine (Sigma-Aldrich) and pseudouridine (TCI Chemicals).

[0165] The mass spectrometer was set to positive ion mode and operated with selected reaction monitoring. The precursor ion m / z was 245.1, and the product ion m / z for uridine was 113.1, while that for pseudouridine was 125.1. Quantification was performed using EIC of the base fragments. Precise mass fractions of the corresponding base fragments were extracted using XCaliburQual Browser and Xcalibur Quan Browser software (Thermo Fisher Scientific) for quantification. Quantification was performed by comparison with a standard curve obtained from pure nucleoside standards from the same batch of samples. The pseudouridine level in the sample was expressed as a percentage of the total uridine content (methylated and unmethylated) and calculated using the following formula: Pseudouridine (%) = 100 × Pseudouridine / [uridine]. Results are as follows. Figure 13 As shown.

[0166] Depend on Figure 12 and Figure 13 It can be seen that the changes in pseudouridine levels between Pus7 knockout mice and loxP mice are consistent with the results of LC-MS / MS detection.

[0167] It will be readily understood by those skilled in the art that the above are merely preferred embodiments of this disclosure and are not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., that can be made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. Application of detection of free modified nucleic acid metabolites in determining nucleic acid molecular modifications in samples.

2. The application according to claim 1, wherein the free modified nucleic acid metabolite is detected by mass spectrometry imaging (MSI). Preferably, the MSI is an ionizing MSI, more preferably a desorption electrospray ionization (DESI) MSI.

3. The application according to claim 1 or 2, wherein the free nucleic acid metabolite may include one or more selected from free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides. Preferably, the free nucleic acid metabolite comprises one or more selected from the group consisting of: methyl adenosine, N6,O2'-dimethyl adenosine, inosine, methyl cytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methyl guanosine, N2,N2-dimethylguanosine, methyl uridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, uridine, pseudouridine, adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine, methyl cytidine 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, uridine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, uridine triphosphate, pseudouridine triphosphate, 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.

4. A method for determining nucleic acid molecule modifications, the method comprising the step of detecting free modified nucleic acid metabolites in a sample.

5. The method according to claim 3, wherein the step of detecting free modified nucleic acid metabolites in the sample includes performing mass spectrometry imaging (MSI) on the sample to obtain MSI data of the free modified nucleic acid metabolites. Preferably, the free nucleic acid metabolite includes one or more selected from free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides. Preferably, the MSI is scanned in positive and / or negative modes. Preferably, the MSI data includes the ionic strength and / or mass-to-charge ratio (m / z) of characteristic adduct ions of free nucleic acid metabolites in the sample, and the sample is preferably in the form of cell / tissue homogenate, cell or tissue slice. Preferably, the ionic strength and / or m / z value of the characteristic adduct of the free nucleic acid metabolite are determined using standards comprising the metabolite. Preferably, the sample is coated on a glass slide. Preferably, the MSI is an ionizing MSI, more preferably a desorption electrospray ionization (DESI) MSI.

6. The method according to claim 3 or 4, wherein the free nucleic acid metabolite may include one or more selected from free modified (deoxy)nucleosides, free (deoxy)nucleosides, free modified (deoxy) nucleotides, and free (deoxy) nucleotides. Preferably, the free nucleic acid metabolite comprises one or more selected from the group consisting of: methyl adenosine, N6,O2'-dimethyl adenosine, inosine, methyl cytidine, 5-hydroxymethyl-2'-deoxycytidine, N4-acetylcytidine, methyl guanosine, N2,N2-dimethylguanosine, methyl uridine, N6-methyl-2'-deoxyadenosine, 2'-deoxyinosine, 5-methyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, uridine, pseudouridine, adenosine, 2'-deoxyguanosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine, methyl cytidine 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, uridine monophosphate, pseudouridine monophosphate, inosine triphosphate, 5-hydroxymethylcytidine triphosphate, uridine triphosphate, pseudouridine triphosphate, 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.

7. The method of claim 4, wherein the adduct ion comprises [M+H] + [M+Na] + [M+K] + [M+NH4] + [M+H-H2O] + [M] + [MH] - [M+Cl] - and [MH-H2O] - One or more of them, Preferably, the adduct ions generated under positive mode scanning include those selected from [M+H]. + [M+Na] + [M+K] + [M+NH4] + [M+H-H2O] + and [M] + One or more of them, Preferably, the adduct ions generated under negative mode scanning include those selected from [MH]. - [M+Cl] - and [MH-H2O] - One or more of them, Preferably, the m / z value range of the positive mode and / or negative mode scan is 0 to 1000, more preferably 20 to 900, and most preferably 30 to 800. Preferably, the m / z value of the characteristic adduct ion is selected from the values ​​in the column of Table 1.

8. The method according to any one of claims 3 to 5, further comprising the step of comparing the obtained MSI data with a control sample, preferably to determine whether the distribution or amount of the free modified nucleic acid metabolite has changed, the change reflecting a change in DNA / RNA molecules.

9. The method according to any one of claims 3 to 6, wherein the method comprises the following steps: 1) Perform mass spectrometry imaging (MSI) on the sample to obtain MSI data under positive and / or negative mode scans; 2) Extract the MSI data to obtain the ionic strength and / or mass-to-charge ratio (m / z) values ​​of the characteristic adduct ions of free nucleic acid metabolites in the sample; and Optionally 3) compare the intensity of the extracted ions with a control, or obtain an MSI image.

10. The application according to any one of claims 1 to 3, or the method according to any one of claims 4 to 9, wherein the sample is a biological sample, preferably obtained from animal or human tissue, body fluid, or cells. Preferably, the sample is obtained from a healthy or diseased subject. Preferably, the sample is applied to a glass slide.

11. A system for performing the method of any one of claims 4 to 10.

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