A method for frozen section preparation for desorption electrospray ionization mass spectrometry imaging (desi-msi) and a method for characterizing spatial distribution of isomeric metabolites in plants or fungi based on ion mobility

Abstract

This invention relates to the field of isomer compound analysis and separation technology, and particularly to a method for preparing frozen sections for DESI-MSI and a method for characterizing the spatial distribution of isomer metabolites in plants or fungi based on ion mobility. This invention first identifies metabolites in tissue extracts using LC-IM-MS, and then characterizes them by sharing drift time... m / z The value is matched with DESI-IM-MSI images. Traditional spatial metabolomics is often limited by relying solely on precise... m / z Annotations cannot distinguish between isomers. In contrast, this invention requires... m / z Simultaneous matching with drift time effectively separates structurally different isomers, thereby improving annotation confidence. The method of this invention was applied to four plants (ginkgo, ginseng, scrophularia, and salvia miltiorrhiza) and one fungus (Ganoderma lucidum), achieving for the first time simultaneous imaging of multiple isomeric metabolites.

Description

Technical Field

[0001] This invention relates to the field of isomer compound analysis and separation technology, and in particular to a method for preparing frozen sections for DESI-MSI and a method for characterizing the spatial distribution of isomer metabolites in plants or fungi based on ion mobility. Background Technology

[0002] Plants and fungi produce a vast array of structurally diverse small-molecule metabolites that are essential for their survival and environmental interactions. This chemical diversity forms the basis of their ecological functions, such as defense, communication, and stress adaptation. However, the distribution of these compounds within tissues is not uniform. Plant ecological adaptability is not merely the sum of its chemical components, but rather determined by the spatial heterogeneity of these compounds within specific tissues, enabling targeted responses to local challenges. Therefore, characterizing the spatial distribution of metabolites is crucial not only for understanding plant ecological strategies but also for ensuring the quality and efficient utilization of agricultural and medicinal products.

[0003] However, this chemical complexity presents significant analytical challenges due to the existence of numerous isomers with nearly identical structures but significantly different biological activities. Ginkgo produces a variety of diterpenoid ginkgolides, including several isomers with different biological activities. Ginkgolide B and ginkgolide J are isomers differing only in the position of the hydroxyl group, but this small structural change leads to a significant difference in platelet-activating factor (PAF) antagonistic activity, with ginkgolide B being 17-22 times more potent than ginkgolide J. This reflects a key principle of plant defense evolution: small molecular modifications can profoundly alter biological activity. Scrophularia produces a series of iridoid ethers that have been reported to possess broad biological activities, possibly related to defense against herbivores or pathogens. Among them, harpagoside and 6'- O -Cinnamyl harpagoside is an isomer, differing only in the position of the cinnamic yl group. While the ecological defense functions of harpagoside (such as inhibition of insect DNA polymerase) have been studied, the specific ecological role of its isomer, 6'-O-cinnamyl harpagoside, remains largely unexplored, representing a significant research gap. This knowledge gap hinders a comprehensive understanding of plant defense chemistry and its interactions within ecosystems. Therefore, characterizing the spatial distribution of key isomers is crucial for understanding plant survival strategies and assessing the quality of medicinal plant materials. However, conventional chemical analytical methods are often insufficient to fully elucidate their true mechanisms of action because they typically require tissue homogenization, thus compromising spatial information.

[0004] Mass spectrometry imaging (MSI) is an advanced molecular imaging method used to visualize the spatial distribution of metabolites in plant and fungal tissues. MSI offers unique advantages in studying metabolite biosynthesis and function. These capabilities make MSI a powerful tool for exploring plant and fungal metabolism, enabling the visualization of multiple compound classes, including lipids, flavonoids, alkaloids, and terpenes, against a natural tissue background. Based on these advantages, MSI has been widely applied in the study of various species, including Ginkgo biloba, ginseng, salvia miltiorrhiza, areca nut, sage, and Ganoderma lucidum. Recent technological innovations have further expanded the capabilities of spatial metabolomics. However, plant and fungal tissues present greater analytical challenges than animal tissues due to the widespread isomerism of their secondary metabolites. Therefore, metabolite annotation in MSI remains a bottleneck: it heavily relies on precise mass spectrometry. m / z Furthermore, precise mass is often insufficient to distinguish isomers and isomeric isotopes from background interference or different adducts. This ambiguity hinders the correlation between chemical structure and spatial distribution, limiting the acquisition of biological insights from MSI data. Although desorption electrospray ionization mass spectrometry (DESI-MSI) facilitates in-situ detection of metabolites, its resolution and sensitivity remain limited by isomer interference. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for preparing frozen sections for DESI-MSI and a method for characterizing the spatial distribution of isomeric metabolites in plants or fungi based on ion mobility. This invention solves the problem that when spatially locating metabolites using MSI, the accuracy of annotation is affected because precise mass alone cannot distinguish isomeric interference.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing frozen sections for DESI-MSI, comprising the following steps: Biological samples were embedded in a carboxymethyl cellulose solution and then frozen to obtain frozen tissue blocks. After attaching tape to the surface of the frozen tissue block, sections were prepared to obtain frozen sections with tape attached. The non-tissue side of the frozen section with adhesive tape is attached and fixed onto a glass slide, the tissue surface is fully exposed, and vacuum dried to obtain a frozen section for DESI-MSI.

[0007] Preferably, the tape is a low-temperature slicing adhesive transfer tape.

[0008] Preferably, the biological sample is at least one of the following: the root, stem, leaf or seed of a plant, and the fruiting body of a fungus.

[0009] Preferably, the thickness of the slice is selected according to the tissue type and physical properties of the biological sample to ensure tissue integrity and preserve spatial information; When the biological sample is a plant root or a fungal fruiting body, the thickness of the slice is 20~40μm; When the biological sample is a plant seed, the thickness of the slice is 30~70μm; When the biological sample is a plant stem, the thickness of the slice is 20~40μm.

[0010] This invention provides the application of frozen sections prepared by the preparation method described above in mass spectrometry imaging.

[0011] This invention provides a method for characterizing the spatial distribution of metabolites in plants or fungi, comprising the following steps: Plants or fungi are freeze-dried and then ground to obtain a dry powder; the dry powder is extracted in a methanol-water solution, centrifuged, the supernatant is collected and filtered to obtain the liquid sample to be tested; LC-IM-MS was used to acquire data from the liquid phase sample to be tested. Peak processing was performed on the acquired data, and finally, metabolite annotation and integration were performed to obtain the results for each metabolite. m / z And the bin value of ion mobility drift time; Prepare plant or fungal samples for DESI-MSI, denoted as the solid phase sample to be tested; DESI-IM-MSI was used to acquire data from the solid-phase sample to be tested. Based on the acquired data, an image was constructed, and the metabolites identified by LC-IM-MS and the ions detected by DESI-IM-MSI were compared using a shared [database / framework]. m / z The correlation and matching criteria were performed with the ion mobility drift time bin value, and the matching criteria were as follows: m / z With a deviation ≤ 5 ppm and a drift time bin deviation ≤ ±5 bins under the same mobility conditions, the spatial distribution of each metabolite is determined on the image constructed by DESI-IM-MSI based on the matching results, and the spatial distribution image of each metabolite is obtained.

[0012] Preferably, the LC-IM-MS data acquisition conditions include: chromatographic separation is performed on a Waters AcquityUPLC HSS T3 column at a column temperature of 40℃; the column specifications are 2.1 mm × 100 mm with a particle size of 1.8 μm; the binary mobile phase consists of phase A and phase B, where phase A is water containing 0.04% acetic acid and phase B is acetonitrile containing 0.04% acetic acid; the gradient elution program is: 0 min 95% A → 10.0 min 5% A → 11.0 min 5% A → 11.1 min 95% A → 15.0 min 95% A; the injection volume is 2 μL; and the flow rate is constant at 0.4 mL / min. Mass spectrometry detection was performed in positive and negative electrospray ionization (ESI) modes using the following optimized parameters: capillary voltage 3.0 kV for positive ion mode and 2.5 kV for negative ion mode; sample cone voltage 20 V; ion source temperature 120 °C; desolvation gas temperature 450 °C; cone gas flow rate 50 L / h; desolvation gas flow rate 800 L / h; and high-resolution HDMS was used. E Full scan mass spectrometry was acquired in this mode, with a scanning range of [missing information]. m / z The range is 50-1200, the scan time is 0.23 s, the low-energy scan collision energy is 6 eV, and the high-energy scan collision energy gradient is 10-40 eV.

[0013] Preferably, the data acquisition conditions for the DESI-IM-MSI include: the DESI spray solvent is a methanol-water solution containing 0.01% formic acid, the volume ratio of methanol to water is 98:2, and the flow rate is 3 μL / min; a complementary acquisition strategy is used for system detection in positive and negative ionization modes: 1) High-resolution HDMS is used for full-scan ion mobility resolution detection; 2) HDMS 2 MS / MS fragmentation for ion mobility resolution; spatial resolution maintained at 100 × 100 μm / pixel, stage movement speed 200 μm / s; ion source temperature stabilized at 120 °C, cone voltage 20 V, full scan at... m / z Data was collected within the range of 50-1200. Ion mobility separation was performed using a nitrogen drift gas. The traveling wave parameters were set to an IMS wave propagation velocity of 375 m / s and a transfer wave velocity of 150 m / s. The number of ion mobility cycles was selected based on the target metabolite. For each species, data from 1 cycle and 10 cycles were evaluated, and the separation conditions that provided better isomer resolution were selected for spatial mapping. The mobility time for 1 cycle was 12 ms, and the mobility time for 10 cycles was 142 ms.

[0014] Preferably, the solid sample to be tested is prepared using the above-described preparation method.

[0015] Preferably, the metabolite annotation integration includes one or more of the following: annotating metabolites through UNIFI library MS / MS spectrum matching, SciFinder, ChemSpider, PubMed database queries, SIRIUS calculation and prediction, and MARS ion mobility data processing.

[0016] This invention provides a method for preparing frozen sections for DESI-MSI. Frozen sectioning is one of the most commonly used methods for DESI sample preparation. However, obtaining high-quality frozen sections of plant tissue remains challenging. Plant cell walls, large intercellular spaces, and high water content make tissues susceptible to mechanical damage during slicing, unfolding, or transfer using anti-roll plates in conventional cryostats. To address these difficulties, this invention employs a combination of carboxymethyl cellulose (CMC) embedding and tape-assisted slicing. CMC has low interference and is the preferred embedding medium for DESI-based plant tissue analysis; however, its hardness and brittleness after freezing still make slicing difficult. This invention utilizes tape-assisted slicing to provide mechanical support during cutting and transfer, thereby maintaining the integrity of brittle tissues. Experimental results show that the frozen sections prepared by this invention have low background interference while retaining metabolite detection, providing a reliable basis for subsequent DESI-IM-MSI analysis of isomeric metabolites.

[0017] This invention provides a method for characterizing the spatial distribution of metabolites in plants or fungi. The invention first identifies metabolites in tissue extracts using liquid chromatography-ion mobility mass spectrometry (LC-IM-MS), and then characterizes them by shared drift time... m / z The value was matched with desorption electrospray ionization ion mobility mass spectrometry imaging (DESI-IM-MSI) images. Traditional space metabolomics is fundamentally limited by its reliance on precision. m / z Annotations cannot distinguish between isomer mixtures. In contrast, this invention requires... m / z Matching the drift time with the annotation time effectively separates structurally different isomers, thereby improving annotation confidence.

[0018] This invention was applied to four plants (ginkgo, ginseng, scrophularia, and salvia miltiorrhiza) and one fungus (Ganoderma lucidum), achieving for the first time simultaneous imaging of multiple isomeric metabolites, including ginkgolide B / J and ginkgolide C / Q. These isoforms exhibited significant tissue-specific distributions in all five species. This invention also corrects misannotations and resolves site isoforms, isotopic compounds, and background interference that conventional MSI cannot distinguish, providing a robust and universal method for isoform-resolved spatial metabolomics in complex plant and fungal matrices. Attached Figure Description

[0019] Figure 1This section evaluates the DESI-MSI sample preparation method. A shows a schematic diagram of the DESI-MSI experimental workflow; B and C compare the histological morphology of Ganoderma lucidum cap (B) and ginseng root (C) sections, prepared using either conventional slide attachment (left) or the CryoJane tape transfer system (right); D and E show two metabolites ([MH]) in Ganoderma lucidum sections prepared using conventional slide attachment and the CryoJane tape system. - m / z 569.27 and [MH] - m / z DESI-MSI ion image of 513.28; F is a comparison of the total ion intensity of Ganoderma lucidum cap slices prepared with and without CryoJane tape, showing representative ions ( m / z (515.3000, 531.2947, and 553.2791) signal levels were similar between the two methods; all ion images were acquired in negative ion mode; Figure 2 Comparison of tissue morphology preparations using conventional glass slides (left image) and CryoJane tape (right image) for sections of Ginkgo seeds, Ginkgo stems, Salvia miltiorrhiza main root, Salvia miltiorrhiza lateral root, and ginseng root; Figure 3 Representative DESI-MSI mass spectra were acquired from a bare glass slide (A) and blank CryoJane tape fixed to the slide (B); all mass spectra were acquired in negative ion mode. Figure 4 To distinguish between ginkgolide B and ginkgolide J by LC-IM-MS and DESI-IM-MSI; where A is the extract ion chromatogram of ginkgo extract ([MH)). - m / z The retention times of ginkgolide B (4.57 min) and ginkgolide J (3.60 min) were determined using standards; B is a fragment spectrum of ginkgolide B and ginkgolide J, obtained by LC-IM-MS on HDMS. E Obtained from real standards during high-energy scanning; C represents the parent ion ([MH)) selected from the corresponding bin sites of each isomer. - m / z DESI-HDMS imaging of Ginkgolide B and Ginkgolide J standards and their 1:1 mixed droplets collected (423.1291); D represents the product ion of Ginkgolide B (…). m / z 349.1282, [M-C2H2O3] - At bin 39 and ginkgolide J product ion ( m / z349.0914, [M-C3H6O2] - DESI-HDMS of standard droplets collected at bin 27 2 Images; separation parameters are described in the Methods section; data sources: (A, B) LC-IM-MS (10 cycles); (C, D) DESI-IM-MSI (10 cycles); Figure 5 To detect and identify isomers in different plant samples using DESI-IM-MSI; where A is a DESI-HDMS ion image of Ginkgo biloba leaves; B is a DESI-HDMS image of Ginkgo biloba roots. 2 Ion images; C represents DESI-HDMS of Ginkgo biloba seeds. 2 Ion image; D represents harpagoside ([MH)) in Scrophularia ningpoensis leaves (PTFE blot). - , m / z Spatial location of 493.1726, bin 73) within leaf veins; E represents tanshinone I ([M+H) in the cortex of *Salvia miltiorrhiza* root (frozen section). + , m / z Spatial localization of 277.0859, bin 28); except for (E) which was acquired in positive ion mode, all ion images were acquired in negative ion mode; all ion images were acquired using 10 cycles of ion mobility separation. Figure 6 Unknown metabolites were discovered and spatially mapped using DESI-IM-MSI and multi-platform analysis; A represents the integrated workflow for metabolite discovery, combining DESI-IM-MSI data with LC-IM-MS identification for spatial annotation; B represents [MH] in Scrophularia ningpoensis leaves. - m / z 463.0891 (bin 13) and m / z Two isomers of 463.0896 (bin 40) were preliminarily identified as quercetin-3- O - Glucoside and 6-hydroxyluteolin-7- O - Glucoside, showing EIC, drift time distribution, and ion images; drift times differ slightly between the two modes (see Methods section); C represents [MH] from Scrophularia ningpoensis leaves. - m / z 345.1196 and m / zTwo isomers of 345.1197 were preliminarily identified as aucubin (bin 27) and harpagoside (detected in fragment form, bin 73), respectively. EIC, drift time distribution, and ion images are shown. The drift times in the ion images correspond to DESI-IM-MSI measurements, which differ slightly from LC-IM-MS values ​​(see Methods section). All ion images were acquired in negative ion mode (HDMS). Data sources: EIC and drift time distribution in (B, C) are from LC-IM-MS; ion images in (B, C) are from DESI-IM-MSI. All ion images were acquired using 10 cycles of ion mobility separation. Figure 7 Tissue-specific distribution of isomeric metabolites analyzed by DESI-IM-MSI; where A is Ginkgo leaf, B is Scrophularia leaf, C is Ganoderma lucidum cap, D is ginseng root, and E is Salvia miltiorrhiza root; all ion images were acquired in negative ion mode; ion mobility separation: (AC) 10 cycles; (D, E) 1 cycle. Detailed Implementation

[0020] This invention provides a method for preparing frozen sections for DESI-MSI, comprising the following steps: Biological samples were embedded in a carboxymethyl cellulose solution and then frozen to obtain frozen tissue blocks. After attaching tape to the surface of the frozen tissue block, sections were prepared to obtain frozen sections with tape attached. The non-tissue side of the frozen section with adhesive tape is attached and fixed onto a glass slide, the tissue surface is fully exposed, and vacuum dried to obtain a frozen section for DESI-MSI.

[0021] This invention involves embedding biological samples in a carboxymethyl cellulose solution and then freezing them to obtain frozen tissue blocks.

[0022] In this invention, the biological sample is preferably at least one of the following: the root, stem, leaf, or seed of a plant, and the fruiting body of a fungus. This invention does not specifically limit the types of plants and fungi; any plant or fungus well-known in the art may be used. In embodiments of this invention, the plant may specifically be ginkgo, ginseng, scrophularia, or salvia miltiorrhiza; the fungus may be ganoderma.

[0023] In this invention, the concentration of the carboxymethyl cellulose solution is preferably 3% (w / v). Preferably, the biological sample is embedded in the carboxymethyl cellulose solution, placed in a silicone mold, and then cryo-coagulated. In this invention, the cryo-coagulation temperature is preferably -80°C.

[0024] After obtaining the frozen tissue block, the present invention attaches adhesive tape to the surface of the frozen tissue block, and then sections it to obtain frozen sections with adhesive tape attached.

[0025] In this invention, the tape is preferably a cryosurfactant transfer tape for cryosectioning, more preferably CryoJane tape; the sectioning is preferably performed using a cryostat; in an embodiment of this invention, a Leica Biosystems CM1950 cryostat is specifically used. The temperature of the frozen tissue block during sectioning is preferably -20°C. In this invention, the thickness of the section is selected according to the tissue type and physical properties of the biological sample to ensure tissue integrity and preserve spatial information; when the biological sample is a plant root or fungal fruiting body, the thickness of the section is 20~40 μm, and in a specific embodiment it can be 20, 25, 30, 35 or 40 μm; when the biological sample is a plant seed, the thickness of the section is 30~70 μm, and in a specific embodiment it can be 30, 40, 50, 60 or 70 μm; when the biological sample is a plant stem, the thickness of the section is 20~40 μm, and in a specific embodiment it can be 20, 25, 30, 35 or 40 μm. This invention optimizes slice thickness based on the physical properties of each tissue type to ensure tissue integrity and preserve spatial information.

[0026] After obtaining frozen sections with adhesive tape, the present invention attaches and fixes the non-tissue surface of the frozen sections with adhesive tape onto a glass slide, fully exposing the tissue surface, and then vacuum-drying to obtain frozen sections for DESI-MSI.

[0027] In this invention, the non-tissue side of the frozen section with adhesive tape is preferably attached and fixed to a glass slide using double-sided adhesive tape. This invention does not impose special requirements on the vacuum drying conditions; any vacuum drying conditions well-known in the art are acceptable. In an embodiment of this invention, the vacuum drying is performed in a desiccator for 10 minutes.

[0028] Conventional frozen sections for DESI-MSI are prepared without tape; instead, the tissue is directly sectioned after freezing and solidification, and then a glass slide is brought close to the tissue section, allowing the tissue section to automatically adhere to the slide, resulting in a frozen section for DESI-MSI. However, plant cell walls, large intercellular spaces, and high water content make tissues prone to mechanical damage during sectioning, unfolding, or transfer using anti-roll plates in conventional cryostats. This invention utilizes tape-assisted sectioning to provide mechanical support during cutting and transfer, thereby maintaining the integrity of brittle tissues. Sections prepared by this method have intact tissue morphology, providing a stable matrix for subsequent analysis. Compared to conventional slide attachment, the CryoJane tape-slide method can successfully section various tissue types that are difficult to process using conventional methods, including Ganoderma lucidum caps and ginseng roots.

[0029] This invention provides the application of frozen sections prepared by the preparation method described above in mass spectrometry imaging.

[0030] This invention provides a method for characterizing the spatial distribution of metabolites in plants or fungi, comprising the following steps: Plants or fungi are freeze-dried and then ground to obtain a dry powder; the dry powder is extracted in a methanol-water solution, centrifuged, the supernatant is collected and filtered to obtain the liquid sample to be tested; LC-IM-MS was used to acquire data from the liquid phase sample to be tested. Peak processing was performed on the acquired data, and finally, metabolite annotation and integration were performed to obtain the results for each metabolite. m / z And the bin value of ion mobility drift time; Prepare plant or fungal samples for DESI-MSI, denoted as the solid phase sample to be tested; DESI-IM-MSI was used to acquire data from the solid-phase sample to be tested. Based on the acquired data, an image was constructed, and the metabolites identified by LC-IM-MS and the ions detected by DESI-IM-MSI were compared using a shared [database / framework]. m / z The correlation and matching criteria were performed with the ion mobility drift time bin value, and the matching criteria were as follows: m / z With a deviation ≤ 5 ppm and a drift time bin deviation ≤ ±5 bins under the same mobility conditions, the spatial distribution of each metabolite is determined on the image constructed by DESI-IM-MSI based on the matching results, and the spatial distribution image of each metabolite is obtained.

[0031] The following is an explanation of LC-IM-MS.

[0032] This invention involves freeze-drying plants or fungi and then grinding them to obtain a dry powder; the dry powder is then extracted in a methanol-water solution, centrifuged, the supernatant is collected and filtered to obtain a liquid sample to be tested.

[0033] In this invention, the volume concentration of methanol in the methanol-water solution is preferably 70%; the extraction temperature is preferably 4°C; and the extraction time is preferably 10 hours. In this invention, the centrifugation speed is preferably 12000 rpm, the centrifugation temperature is preferably 4°C, and the centrifugation time is preferably 10 minutes. In this invention, the pore size of the filter membrane used for filtration is preferably 0.22 μm.

[0034] After obtaining the liquid phase sample to be tested, this invention uses LC-IM-MS to acquire data from the sample, performs peak processing on the acquired data, and finally performs metabolite annotation and integration, thereby obtaining the data of each metabolite. m / z And the bin value of ion mobility drift time.

[0035] In this invention, the preferred conditions for LC-IM-MS data acquisition include: chromatographic separation is performed on a WatersAcquity UPLC HSS T3 column at a column temperature of 40°C; the column specifications are 2.1 mm × 100 mm with a particle size of 1.8 μm; the binary mobile phase consists of phase A and phase B, where phase A is water containing 0.04% acetic acid and phase B is acetonitrile containing 0.04% acetic acid; the gradient elution program is: 0 min 95% A → 10.0 min 5% A → 11.0 min 5% A → 11.1 min 95% A → 15.0 min 95% A; the injection volume is 2 μL; and the flow rate is constant at 0.4 mL / min. Mass spectrometry detection was performed in positive and negative electrospray ionization (ESI) modes using the following optimized parameters: capillary voltage 3.0 kV for positive ion mode and 2.5 kV for negative ion mode; sample cone voltage 20 V; ion source temperature 120 °C; desolvation gas temperature 450 °C; cone gas flow rate 50 L / h; desolvation gas flow rate 800 L / h; and high-resolution HDMS was used. E Full scan mass spectrometry was acquired in this mode, with a scanning range of [missing information]. m / z The range is 50-1200, the scan time is 0.23 s, the low-energy scan collision energy is 6 eV, and the high-energy scan collision energy gradient is 10-40 eV.

[0036] After data acquisition, the present invention performs peak processing on the obtained data. The present invention does not impose any special limitations on the peak processing process, and any peak processing process well known in the art can be used, such as using QI software (v3.0, Waters) for peak identification, matching, alignment, extraction, integration and normalization.

[0037] After peak processing, this invention performs metabolite annotation and integration.

[0038] In this invention, the metabolite annotation integration preferably includes one or more of the following methods: annotating metabolites by matching MS / MS spectra from the UNIFI library, querying SciFinder, ChemSpider, and PubMed databases, calculating and predicting using SIRIUS, and processing ion mobility data using MARS.

[0039] This invention utilizes LC-IM-MS to separate and identify metabolites, and these identified ions are the diagnostic ions for subsequent DESI-IM-MSI differentiation.

[0040] The following is an explanation of DESI-IM-MSI.

[0041] In this invention, plant or fungal samples for DESI-MSI are first prepared and denoted as the solid phase sample to be tested.

[0042] In this invention, the solid sample to be tested is preferably prepared using the preparation method described above. When the sample is a leaf, frozen section samples can be prepared using the above preparation method, or the well-known metabolite blotting method can be used to prepare the sample to be tested.

[0043] After obtaining the solid sample to be tested, the present invention uses DESI-IM-MSI to collect data on the solid sample and constructs an image based on the collected data.

[0044] In this invention, the preferred conditions for data acquisition using the DESI-IM-MSI method include: the DESI spray solvent is a methanol-water solution containing 0.01% formic acid, the volume ratio of methanol to formic acid is 98:2, and the flow rate is 3 μL / min; a complementary acquisition strategy is used for system detection in both positive and negative ionization modes: 1) High-resolution HDMS is used for full-scan ion mobility resolution detection; 2) HDMS 2 MS / MS fragmentation for ion mobility resolution; spatial resolution maintained at 100 × 100 μm / pixel, stage movement speed 200 μm / s; ion source temperature stabilized at 120 °C, cone voltage 20 V, full mass spectrometry at... m / z Data was collected within the range of 50-1200. Ion mobility separation was performed using nitrogen drift gas. The traveling wave parameters were set to IMS wave propagation velocity of 375 m / s and transfer wave velocity of 150 m / s. The number of ion mobility cycles (1 cycle, 12 ms; 10 cycles, 142 ms) was selected based on the target metabolite. For samples for which data from 1 cycle and 10 cycles were collected simultaneously, the two conditions were evaluated and the isomer resolution was selected for spatial mapping.

[0045] Finally, this invention integrates LC-IM-MS and DESI-IM-MSI annotations across platforms. Specifically, it integrates metabolites identified by LC-IM-MS and ions detected by DESI-IM-MSI through a shared annotation mechanism. m / z The correlation and matching criteria were performed with the ion mobility drift time bin value, and the matching criteria were as follows: m / z With a deviation ≤ 5 ppm and a bin deviation ≤ ±5 bins under the same mobility conditions, the spatial distribution of each metabolite is determined on the image constructed by DESI-IM-MSI based on the matching results, resulting in a spatial distribution image of each metabolite. This invention utilizes shared drift time... m / z The value associates LC-IM-MS identification with DESI-IM-MSI imaging. This strategy can utilize retention time, drift time and MS / MS data for orthogonal verification, which solves the annotation ambiguity inherent in traditional MSI.

[0046] The following detailed description, in conjunction with embodiments, of the method for preparing frozen sections for DESI-MSI and the method for characterizing the spatial distribution of metabolites in plants or fungi provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0047] The chemicals and reagents used in the following examples are as follows: HPLC-grade acetonitrile, acetic acid, and methanol were purchased from Merck (Darmstadt, Germany). Water was purified using a MilliQ ULTRA purification system (Millipore, Vimodrone, Italy). The internal standard lidocaine (137-58-6) used in this invention was purchased from Aladdin Technology (Shanghai, China). Ginkgolide B (15291-77-7), Ginkgolide C (15291-76-6), and Ginkgolide J (107438-79-9) reference standards (>99%, quantitative grade) were purchased from Aladdin Technology (Shanghai, China). Tanshinone I (568-73-0) and harpagoside (19210-12-9) reference standards (>98%, quantitative grade) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). All standards were stored at -80°C protected from light. All other reagents were of analytical grade. Sodium carboxymethyl cellulose (CMC) was purchased from Aladdin Technology (Shanghai, China). The porous polytetrafluoroethylene (PTFE) membrane, with a thickness of 1.6 mm, a porosity of 36%, and a pore size of 7 μm, was purchased from Berghof Group (Eningen, Germany).

[0048] The sample collection process is as follows: Leaves and roots of two-year-old Ginkgo seedlings were collected in March, and seeds of twenty-year-old trees were collected in September, both from Zhejiang University's Zijingang Campus. Scrophularia leaves were collected from two-year-old seedlings at Zhejiang University. Ganoderma lucidum caps (umbrellas) were obtained from the Large Fungal Specimen Museum of Zhejiang University. Ginseng roots were collected from Changchun. Salvia miltiorrhiza roots were collected from two-year-old seedlings at Zhejiang University of Science and Technology. Samples used for non-targeted metabolomics were transported to the laboratory in liquid nitrogen and stored at -80°C, while samples used for spatial metabolomics were flash-frozen in liquid nitrogen and processed immediately. Tissue samples for each species were derived from a single representative plant or fungal specimen. All spatial distributions reported in this invention are derived from these samples.

[0049] The characterization methods in the following embodiments are as follows: Sample preparation for DESI-MSI analysis: Frozen fresh roots, seeds, and caps were embedded in 3% (w / v) CMC solution and placed in a silicone mold (2×2×2 cm). The mold was then transferred to a -80°C freezer for overnight solidification. Serial sections were cut from the frozen tissue blocks at -20°C using a cryostat (Leica Biosystems, CM1950) with a tape window (Leica Biosystems). The thickness of root and cap sections was 20 μm, and the thickness of seed sections was 50 μm. The section thickness was optimized according to the physical properties of each tissue type to ensure tissue integrity and preserve spatial information. The non-tissue side of the cryostat was directly attached to a glass slide with double-sided tape and vacuum dried in a desiccator for 10 min before DESI-MSI analysis. For PTFE-based metabolite blots, fresh leaves were sandwiched between two PTFE membranes. The assemblies were pressed under constant pressure (5 MPa) for 60 s using a hydraulic press to promote metabolite transfer to the membrane interlayer. Only the far-axis surface in contact with the membrane was selected for subsequent DESI-IM-MSI analysis.

[0050] Sample preparation for LC-IM-MS analysis: The materials were lyophilized using a vacuum freeze dryer. The resulting lyophilized product was then homogenized in a ball mill (30 Hz, 1 min). 100 mg of the dried powder was extracted with 1.0 mL of 70% methanol aqueous solution (v / v) at 4 °C for 10 h. After extraction, the samples were centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was collected and filtered through a 0.22 μm organic filter into vials. Lidocaine was used as an internal standard.

[0051] The data acquisition conditions for LC-IM-MS are as follows: Metabolite analysis was performed using an ACQUITY UPLC system (Waters, Milford, MA, USA) coupled with a SELECT SERIES Cyclic IMS mass spectrometer. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm particle size) at 40 °C. The binary mobile phase consisted of phase A (water containing 0.04% acetic acid) and phase B (acetonitrile containing 0.04% acetic acid), with the following gradient program: initial (0 min) 95% A, linearly decreasing to 5% A at 10.0 min, holding at 5% A to 11.0 min, rapidly increasing to 95% A at 11.1 min, and finally holding at 95% A for column reequilibration until 15.0 min. The injection volume was 2 μL, and the flow rate was kept constant at 0.4 mL / min throughout the analysis.

[0052] Mass spectrometry detection was performed in electrospray ionization (ESI) modes with the following optimized parameters: capillary voltage 3.0 kV for positive ion mode and 2.5 kV for negative ion mode; sample cone voltage 20 V; ion source temperature 120 °C; desolvation gas temperature 450 °C; cone gas flow rate 50 L / h; desolvation gas flow rate 800 L / h. High-resolution MSE (HDMS) was also used. E Full scan mass spectrometry is acquired in ) mode. Full scan mass spectrometry is in m / z Data was acquired within the range of 50-1200, with a scan time of 0.23 s. The collision energy for low-energy scans was 6 eV, and the collision energy gradient for high-energy scans was 10-40 eV. Before analysis, the mass axis was calibrated daily using 0.5 mM sodium formate solution. Simultaneously, during data acquisition, real-time mass calibration was performed by continuous infusion of leucine enkephalin (200 pg / μL) via a lock spray.

[0053] The DESI-IM-MSI analysis conditions are as follows: All DESI-IM-MSI analyses were performed on a high-resolution SELECT SERIES Cyclic IMS platform (Waters Corporation, Milford, MA, USA) equipped with a dedicated DESI ion source (located at the mass spectrometer inlet). Tissue sections were systematically detected using a complementary acquisition strategy in both positive and negative ionization modes: 1) High-resolution HDMS was used for full-scan ion mobility resolution detection; 2) HDMS 2 MS / MS fragmentation was used for ion mobility resolution. Key instrument parameters were set as follows: spatial resolution maintained at 100 × 100 μm / pixel, stage movement speed 200 μm / s. Spatial resolution was set to 100 μm (stage step size) to balance acquisition time with tissue section size. Ion source temperature was stabilized at 120℃, cone voltage 20 V, and full mass spectrometry was performed at... m / z Data were collected within the range of 50-1200. The optimized electrospray solvent was HPLC-grade methanol (98:2 v / v) containing 0.01% formic acid, with 200 pg / μL of leucine enkephalin added for continuous mass calibration, delivered at 3 μL / min using a Harvard Pump 11 Elite syringe pump (Harvard Apparatus, Holliston, MA). Ion mobility separation was performed using a nitrogen drift gas, with traveling wave parameters set to an IMS wave propagation velocity of 375 m / s and a transfer wave velocity of 150 m / s. These conditions were suitable for collision cross-section measurements in nitrogen. The number of ion mobility cycles (1 cycle, 12 ms; 10 cycles, 142 ms) was selected based on the target metabolite; for samples with both 1-cycle and 10-cycle data were collected simultaneously, the two conditions were evaluated, and the one with better isomer resolution was selected for spatial mapping.

[0054] Under 10 cycles (142 ms) conditions, the typical full width at half maximum (FWHM) for representative metabolites is approximately 8 bins; under 1 cycle conditions, the typical FWHM is approximately 5 bins. Drift times are reported as instrument-specific bins rather than CCS values ​​because the multi-cycle separation results in analytes and calibration ions undergoing different number of passes in the circulating mobility cell, making it impossible to reliably establish a uniform CCS calibration profile; reporting drift times by bin is standard practice for multi-cycle IM. All drift time comparisons between LC-IM-MS and DESI-IM-MSI were performed under the same mobility conditions. A slight DT shift was observed between the two modes due to different ionization conditions; metabolite identity was validated using the complete cube rather than just precise DT matching. When cross-platform referencing, when m / z A match was considered achieved when the deviation was within 5 ppm and the drift time was within ±5 bins. Ion images were displayed using individually optimized intensity thresholds to best represent spatial distribution; therefore, signal intensities between different ion images cannot be directly compared. In the blank control experiment, blank slides and blank CryoJane tapes attached to the slides were analyzed under the same DESI-IM-MSI conditions. All mass spectrometry data were acquired using MassLynx software (v4.2, Waters Corporation) in both positive and negative ionization modes. Subsequent raw data processing, metabolite visualization, and spatial distribution analysis were performed using HDI Imaging Software and DriftScope (Waters Corporation), and spectral demixing was applied for image generation.

[0055] Example 1 Sample preparation for DESI-MSI analysis: according to Figure 1 As shown in Figure A, frozen fresh roots, seeds, and caps were embedded in 3% (w / v) CMC solution and placed in a silicone mold (2×2×2 cm). The mold was then transferred to a -80°C freezer for overnight solidification. Serial sections were cut from the frozen tissue blocks at -20°C using a cryostat (Leica Biosystems, CM1950) with a tape window (Leica Biosystems). The root and cap sections were 20 μm thick, and the seed sections were 50 μm thick. The non-tissue side of the cryostat was directly attached to a glass slide using double-sided tape, and the slides were vacuum-dried in a desiccator for 10 min before DESI-MSI analysis to obtain samples for DESI-MSI analysis.

[0056] The sections prepared in Example 1 have complete tissue morphology, see Figure 1 B and C. Among them,Figure 1 Image B shows a comparison of the morphology of Ganoderma lucidum cap sections prepared using conventional glass slide attachment (left image) and CryoJane tape (right image). Figure 1 The comparison of tissue morphology of ginseng root sections prepared using conventional glass slides (left image) and CryoJane tape (right image) clearly shows that the frozen sections prepared using the tape in this invention have more intact tissue.

[0057] In addition, using the above method, Ginkgo seeds, Ginkgo stems, Salvia miltiorrhiza main root, Salvia miltiorrhiza lateral root, and ginseng root were prepared into frozen sections, and their histomorphology was compared with that of conventionally mounted glass slides. The results are shown in […]. Figure 2 The left image shows a conventionally mounted glass slide, while the right image shows a slide mounted using CryoJane tape. Figure 2 It is evident that the frozen tissue sections prepared using adhesive tape in this invention are more complete.

[0058] DESI-MSI analysis performed in negative ion mode revealed the easy detection of a variety of representative metabolites in the tape-attached sections. Specifically, the structurally characterized triterpenoid compound ganoderic leucine F ([MH)) was also detected. - m / z 569.2756) and ganoderic acid B ([MH] - m / z 513.2858) can be clearly detected in both preparation methods (see Figure 1 (D and E). Spatial distribution patterns further revealed that ganoderic acids were mainly located in the outermost layer of the Ganoderma lucidum cap, consistent with previous reports. To further assess the potential interference of the tape, blank control spectra were collected from blank slides and blank CryoJane tape attached to the slides. The results are shown in […]. Figure 3 The tape only introduces two background signals. m / z 163.1124 and m / z 339.2324, both of which are related to the target metabolites annotated in this invention. m / z The values ​​do not overlap.

[0059] In addition, to verify the reliability of the tape-assisted method, repeated experiments were conducted using different Ganoderma lucidum cap specimens, with and without CryoJane. Direct comparison showed that the total ionic strength of the two preparation methods was roughly equivalent, with representative ions (such as...) m / z 515.3000, 531.2947, and 553.2791) exhibit similar signal levels ( Figure 1The results (F) indicate that the tape system does not cause significant signal loss or enhancement. These results demonstrate that the tape-assisted protocol, systematically evaluated on the DESI-IM-MSI platform, can produce high-quality frozen sections while preserving metabolite detection, providing a reliable basis for subsequent DESI-IM-MSI analysis of isomeric metabolites.

[0060] Example 2 This embodiment uses isomer standards ginkgolide B and ginkgolide J (C 20 H 24 O 11 [MH] - , m / z The separation performance of LC-IM-MS was evaluated (423.1291). Baseline separation of the two compounds was achieved by liquid chromatography, with retention times of 4.57 min and 3.60 min, respectively. Their drift times also differed, with ginkgolide J drifting at bin 27 and ginkgolide B at bin 40. Figure 4 (See section A). Subsequently, the same LC-IM-MS method was used to analyze the Ginkgo biloba leaf extract. Compared with the standard, the extracted ion chromatogram of the plant sample contained multiple peaks at the same precursor ion, reflecting greater complexity and the presence of coexisting isomers in the natural matrix. Figure 4 (B). The complexity of natural extracts underscores the necessity of obtaining reliable structural information prior to imaging. By comparing the MS / MS spectra of ginkgolide B and J, this invention identified their respective unique fragment ions, which can serve as diagnostic ions for differentiation in subsequent MSI.

[0061] After establishing LC-IM-MS separation, the spatial resolving power of DESI-IM-MSI was evaluated. Standard solutions of ginkgolide B, ginkgolide J, and their 1:1 mixture were spotted onto a glass slide. In HDMS mode, which depends on MS1 ​​and drift time, ginkgolide B and the mixed droplets... m / z At positions 423.1291 and 37, ginkgolide J was specifically detected, while no signal was detected in ginkgolide J and the mixed droplets. m / z However, a unique signal was observed at drift time bin 24, where no signal was found for ginkgolide B. Figure 4 (C). These results indicate that the MS1 binding drift time is sufficient to distinguish these isomers.

[0062] To further improve specificity, this invention utilizes HDMS with the aforementioned diagnostic ions. 2 Pattern. Ginkgolide B produces characteristic ions. m / z 349.1278 ([M-C2H2O3]) -Ginkgolide J produces unique fragments. m / z 349.0896 ([M-C3H6O2]) - Mass spectrometry imaging based on these characteristic fragments and their corresponding drift times (ginkgolides B are...). m / z 349.1282, bin 39; Ginkgolide J is m / z 349.0914, bin 27), signals are generated only from the correct isomer standards and mixtures ( Figure 4 (D). These results indicate that DESI-IM-MSI is effective in HDMS and HDMS. 2 In all detection modes, it can distinguish isomeric metabolites with high specificity under the test conditions.

[0063] Example 3 To verify the practical applicability of the method of this invention in biological samples, this embodiment applies the method of this invention to Ginkgo biloba leaves, a species that presents a challenge due to the high complexity of isomeric ginkgolides. Using DESI-HDMS mode, this invention distinguishes ginkgolide J (… m / z 423.1294, bin 25, [MH] - ) and its isomers ginkgolide B, M, F and P ( m / z 423.1294, bin 37, [MH] - These isomers are distributed along the leaf veins, with a weak signal at the outermost leaf margin. Figure 5 (A). Furthermore, this embodiment analyzed ginkgolide C (…). m / z 439.1257, bin 46, [MH] - ) and Ginkgolide Q ( m / z 439.1299, bin 22, [MH] - Both are mainly located on the leaf margin epidermis.

[0064] Previous studies have reported that ginkgolide A and other isomers (B, J, M) are distributed in the epidermis and mesophyll of Ginkgo leaves. Spatial mapping of ginkgolide C and its isomers in this invention demonstrates that DESI-HDMS can achieve more precise localization and provides additional spatial information on ginkgolide distribution. To further enhance isomer differentiation, this invention utilizes HDMS... 2 This method was applied to frozen sections of Ginkgo biloba roots. Analysis of MS / MS fragments in Ginkgo biloba LC-IM-MS data revealed the presence of characteristic fragment ions. Combined with MS1 and MS2 analyses, supplemented by drift time, ginkgolides J (…) were differentiated. m / z 423.1316, bin 27, [MH] - ) and its isomers ginkgolide B, M and F (characteristic fragment ions)m / z 367.1410, bin 41, [MH] - These compounds are primarily located in the phloem of Ginkgo biloba roots. Figure 5 (B). Building upon previous detections of ginkgolide isomers (B, J, M) in the root cortex and phloem, this invention further elucidates their spatial distribution, achieving specific imaging of ginkgolide J, unaffected by coexisting isomers. Similarly, analysis of ginkgo seeds further supports the applicability of this method. Figure 5 (C)

[0065] Example 4 The method of this invention was applied to the analysis of Scrophularia ningpoensis leaves. Scrophularia ningpoensis accumulates a large amount of iridoid glycosides, such as harpagoside and 6'- O - Cinnamyl harpagoside (C 24 H 30 O 11 [MH] - , m / z 493.1716). For Scrophularia, PTFE-imprinted leaves were analyzed. In LC-IM-MS analysis, harpagoside and its isomer 6'- O - Cinnamyl harpagoside exhibited different drift times (bin 73 and bin 60), achieving baseline separation ( Figure 5 (D). By selecting a specific drift time (bin73) for harpagoside, DESI-IM-MSI revealed that it is mainly enriched along the mid-vein, with significantly weaker signals along the lateral veins, and is not affected by its isomers.

[0066] Example 5 The method of this invention was applied to frozen slices of Salvia miltiorrhiza root. Salvia miltiorrhiza is characterized by a high concentration of tanshinones, including tanshinone I and isotanshinone I (C...). 18 H 12 O3, [M+H] + , m / z 277.0859). For example... Figure 5 As shown in E, tanshinone I ( m / z 277.0859, bin 28, [M+H] + The cells were primarily located in the root cortex, a distribution consistent with previous reports. These results demonstrate that a multidimensional validation strategy integrating drift-time separation, characteristic fragment ion detection, and spatial distribution validation in DESI-IM-MSI can support more reliable isoform annotation in plants.

[0067] Example 6 This embodiment correlates the metabolites identified by LC-IM-MS in tissue extracts with their spatial distribution in DESI-IM-MSI ( Figure 6(A) Multidimensional data, such as MS1, MS2, retention time, and drift time, were collected using non-targeted metabolomics. These data were then analyzed using tools from multiple platforms (QI, MARS, DriftScope, and SIRIUS) to enhance annotation robustness. Finally, the imaging data were analyzed... m / z Cross-referencing bin values ​​with non-targeted metabolomics results allows for the annotation of metabolites.

[0068] The method of this invention was applied to Scrophularia ningpoensis leaves, and two pairs of isomers were distinguished by ion mobility separation and multi-platform spectral analysis. First, in m / z 463.0882 [MH] - ( Figure 6 At position B), the extracted ion chromatogram showed two distinct peaks (peak 1, 2.93 min; peak 2, 3.23 min), with significantly different drift times (peak 1 at bin 9, peak 2 at bin 35). Their MS / MS spectra shared characteristic fragment ions consistent with those of pentoxyflavonoid glycosides, but the specific aglycones and glycosylation sites may differ. Based on literature and database comparisons, they are tentatively identified as quercetin-3- O - Glucoside and 6-hydroxyluteolin-7- O - Glucoside. Significant differences in drift time allowed these two isomers to be clearly distinguished despite their similar precise mass and fragmentation patterns. Their tissue-specific spatial distribution was further visualized using DESI-IM-MSI.

[0069] The value of ion mobility in resolving isomer interference m / z This is further reflected at point 345.1191 ( Figure 6 (C). Both chromatographic peaks (peak 3, 1.86 min; peak 4, 4.43 min) showed this ion, and the MS / MS fragments were similar, leading to spectral matching and labeling both as aucubin. However, their different drift times (peak 3 at bin 27, peak 4 at bin 73) indicated that they corresponded to different compounds. Careful examination of the MS / MS spectra revealed a distinguishing fragmentation pattern: peak 4 showed characteristic harpagoside fragmentation, with the parent ion... m / z 493.1690 Loss of cinnamon yl group produces m / z 345.1161, followed by fragmentation. m / z 165.0545 and 147.0450 (cinnamic acid derivatives). In contrast, peak 3 exhibits typical free monosaccharide characteristics, with a shorter drift time reflecting its more compact gas-phase structure. Based on this integrated evidence, including drift time differences, precise mass, and characteristic fragmentation, peak 4 was designated as harpagoside (detected in its fragmented form), and peak 3 as aucubin (supported by MS / MS matching). This makes harpagoside (detected through its fragments)... m / zSpecific imaging along the midrib (345.1197, bin 73) becomes possible, unaffected by aucubin or other isomers. Figure 6 (C). This distribution is consistent with... Figure 5 The results observed in D were consistent. In contrast, aucubin was primarily localized in the lateral vein network, indicating a distinct tissue-specific distribution pattern between the two compounds.

[0070] Example 7 This invention employs the DESI-IM-MSI strategy to resolve the spatial distribution of isomeric metabolites in five medicinal species (Ginkgo biloba, Scrophularia ningpoensis, Ganoderma lucidum, ginseng, and Salvia miltiorrhiza). This is the first time that isomeric spatial resolution mapping has been performed across multiple medicinal species, including gymnosperms, angiosperms, and fungi. The following spatial patterns were observed in the analyzed samples.

[0071] In ginkgo leaves, two types are tentatively identified as hydroxylated ginkgolic acid C17:1 ( m / z 389.2691, bin 35, [MH] - ) and putative phytosterol derivatives ( m / z 389.2692, bin 18, [MH] - The isomers of ) are separated based on drift time, although they m / z They are the same, but show different spatial distributions. Figure 7 (A). Specifically, hydroxylated ginkgolic acid C17:1 was observed as punctate enrichment along leaf veins, mainly located at vein branching points, while the latter was mainly detected at leaf margins. Furthermore, both... m / z Similar metabolites, oxidized ginkgolic acid C17:1 isomer 1 ( m / z 403.2485, bin 23, [MH] - ) and oxidized ginkgolic acid C17:1 isomer 2 ( m / z 403.2490, bin 36, [MH] - All were observed to accumulate at the outermost leaf margin. These results indicate that ion mobility can distinguish between ions with the same... m / z Structurally similar metabolites.

[0072] Similarly, in Scrophularia leaves, the two pairs of phenolic glycoside isomers (common characteristic components of this medicinal plant) exhibit similar spatial distributions. The isomers of verbaspinoside ( m / z 637.2161, bin 81, [MH] - ) and another isomer ( m / z 637.2142, bin 70, [MH] -Both were enriched at the base of the leaf veins, although their drift times differed slightly. Another pair of iridoid glycosides, caffeoyl-substituted aucubin derivatives ( m / z 653.2104, bin 27, [MH] - ) and saccatoside ( m / z 653.2106, bin 15, [MH] - Both compounds exhibited similar localization patterns, accumulating primarily at the base of leaf veins. Figure 7 (B)

[0073] In the cap of Ganoderma lucidum, the isomer of ganoderic acid R ( m / z 531.2976, bin 11, [MH] - ) and Ganoderma eta ( m / z 531.2978, bin 26, [MH] - Both are characteristic triterpenoids, exhibiting highly similar spatial distributions, mainly enriched in the outermost shell, although their signal intensities differ. Figure 7 In contrast to these triterpenoids, other compound classes exhibit distinct spatial patterns. For example, two isomeric fatty acids were provisionally identified as 9,10,13-trihydroxyoctadecadienoic acid (C) based on database matching. m / z 327.2180, bin 18, [MH] - ) and malyngic acid ( m / z 327.2182, bin 26, [MH] - However, the available MS / MS fragmentation data is insufficient to confidently specify its location heterogeneity.

[0074] In ginseng root slices, ion mobility effectively resolved two pairs of isomers that could not be distinguished by mass alone. The first pair is tentatively identified as arginine isomers ( m / z 173.1034, bin 16 vs. bin 25, [MH] - Both are confined to the outermost root layer and have similar spatial distributions. The second pair is tentatively identified as positional isomers of melitriose ( m / z 539.1380, bin 56; m / z 539.1390, bin 61, [M+Cl] - They also exhibit a similar localization pattern, distributed in the root peripheral tissues ( Figure 7 (D). These results demonstrate the practicality of ion mobility in resolving root tissue heterogeneity.

[0075] In the root of Salvia miltiorrhiza, tetrasaccharide isomer 1 ( m / z665.2131, bin 46, [MH] - The distribution of ) exhibits a distinct bicyclic pattern, with signals present in both the vascular cylinder (xylem) and the outermost cortex. In contrast, tetrasaccharide isomer 2 ( m / z 665.2132, bin 50, [MH] - The observation showed that the specific location was in the outermost layer of the cortex. Figure 7 In addition, phytic acid (E). m / z 315.1948, bin 42, [MH] - It showed significant enrichment in the outermost cortical layer. m / z 315.1946 (bin 46, [MH]) - Iso- and ectopic ions were identified as background interference because they did not exhibit tissue-specific patterns. These results demonstrate the effectiveness of ion mobility in distinguishing background interference from the true signal, thereby improving the accuracy of spatial distribution analysis.

[0076] As can be seen from the above embodiments, the present invention achieves this by sharing drift time and time. m / z This invention correlates LC-IM-MS identification with DESI-IM-MSI imaging. This method utilizes retention time, drift time, and MS / MS data for orthogonal validation, resolving the inherent annotation ambiguities of traditional MSI. After validation in five medicinal species, this strategy achieved, for the first time, simultaneous imaging of ginkgolide isomers in plants and, for the first time, isomer-resolved DESI-IM-MSI in fungi. It resolved multiple isomeric metabolites with different tissue-specific distributions in each species, corrected misannotations, and distinguished positional isomers, isotopic compounds, and background interference. This invention provides a robust and universal method for isomer-resolved spatial metabolomics, which is expected to support quality assessment, elucidation of biosynthetic pathways, and the rational utilization of medicinal plant and fungal resources.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing frozen sections for DESI-MSI, characterized in that, Includes the following steps: Biological samples were embedded in a carboxymethyl cellulose solution and then frozen to obtain frozen tissue blocks. After attaching tape to the surface of the frozen tissue block, sections were prepared to obtain frozen sections with tape attached. The non-tissue side of the frozen section with adhesive tape is attached and fixed onto a glass slide, the tissue surface is fully exposed, and vacuum dried to obtain a frozen section for DESI-MSI.

2. The preparation method according to claim 1, characterized in that, The tape is a low-temperature slicing adhesive transfer tape.

3. The preparation method according to claim 1, characterized in that, The biological sample is at least one of the following: the root, stem, leaf, or seed of a plant, and the fruiting body of a fungus.

4. The preparation method according to claim 3, characterized in that, The thickness of the slices is selected based on the tissue type and physical properties of the biological sample to ensure tissue integrity and preserve spatial information; When the biological sample is a plant root or a fungal fruiting body, the thickness of the slice is 20~40μm; When the biological sample is a plant seed, the thickness of the slice is 30~70μm; When the biological sample is a plant stem, the thickness of the slice is 20~40μm.

5. The application of frozen sections prepared by the preparation method according to any one of claims 1 to 4 in mass spectrometry imaging.

6. A method for characterizing the spatial distribution of metabolites in plants or fungi, characterized in that, Includes the following steps: Plants or fungi are freeze-dried and then ground to obtain a dry powder; the dry powder is extracted in a methanol-water solution, centrifuged, the supernatant is collected and filtered to obtain the liquid sample to be tested; LC-IM-MS was used to acquire data from the liquid phase sample to be tested. Peak processing was performed on the acquired data, and finally, metabolite annotation and integration were performed to obtain the results for each metabolite. m / z And the bin value of ion mobility drift time; Prepare plant or fungal samples for DESI-MSI, denoted as the solid phase sample to be tested; DESI-IM-MSI was used to acquire data from the solid-phase sample to be tested. Based on the acquired data, an image was constructed, and the metabolites identified by LC-IM-MS and the ions detected by DESI-IM-MSI were compared using a shared [database / framework]. m / z The correlation and matching criteria were performed with the ion mobility drift time bin value, and the matching criteria were as follows: m / z With a deviation ≤ 5 ppm and a drift time bin deviation ≤ ±5 bins under the same mobility conditions, the spatial distribution of each metabolite is determined on the image constructed by DESI-IM-MSI based on the matching results, and the spatial distribution image of each metabolite is obtained.

7. The method according to claim 6, characterized in that, The LC-IM-MS data acquisition conditions included: chromatographic separation was performed on a Waters Acquity UPLC HSS T3 column at a column temperature of 40℃; the column specifications were 2.1 mm × 100 mm with a particle size of 1.8 μm; the binary mobile phase consisted of phase A and phase B, where phase A was water containing 0.04% acetic acid and phase B was acetonitrile containing 0.04% acetic acid; the gradient elution program was: 0 min 95% A → 10.0 min 5% A → 11.0 min 5% A → 11.1 min 95% A → 15.0 min 95% A; the injection volume was 2 μL, and the flow rate was constant at 0.4 mL / min. Mass spectrometry detection was performed in positive and negative electrospray ionization (ESI) modes using the following optimized parameters: capillary voltage 3.0 kV for positive ion mode and 2.5 kV for negative ion mode; sample cone voltage 20 V; ion source temperature 120 °C; desolvation gas temperature 450 °C; cone gas flow rate 50 L / h; desolvation gas flow rate 800 L / h; and high-resolution HDMS was used. E Full scan mass spectrometry was acquired in this mode, with a scanning range of [missing information]. m / z The range is 50-1200, the scan time is 0.23 s, the low-energy scan collision energy is 6 eV, and the high-energy scan collision energy gradient is 10-40 eV.

8. The method according to claim 6, characterized in that, The data acquisition conditions for the DESI-IM-MSI include: the DESI spray solvent is a methanol-water solution containing 0.01% formic acid, the volume ratio of methanol to water is 98:2, and the flow rate is 3 μL / min; a complementary acquisition strategy is used for system detection in both positive and negative ionization modes: 1) High-resolution HDMS is used for full-scan ion mobility resolution detection; 2) HDMS 2 MS / MS fragmentation for ion mobility resolution; spatial resolution maintained at 100 × 100 μm / pixel, stage movement speed 200 μm / s; ion source temperature stabilized at 120 °C, cone voltage 20 V, full scan at... m / z Data was collected within the range of 50-1200. Ion mobility separation was performed using a nitrogen drift gas. The traveling wave parameters were set to an IMS wave propagation velocity of 375 m / s and a transfer wave velocity of 150 m / s. The number of ion mobility cycles was selected based on the target metabolite. For each species, data from 1 cycle and 10 cycles were evaluated, and the separation conditions that provided better isomer resolution were selected for spatial mapping. The mobility time for 1 cycle was 12 ms, and the mobility time for 10 cycles was 142 ms.

9. The method according to claim 6, characterized in that, The solid sample to be tested was prepared using the preparation method described in any one of claims 1 to 4.

10. The method according to claim 6, characterized in that, The metabolite annotation integration includes one or more of the following methods to annotate metabolites: matching MS / MS spectra from the UNIFI library, querying SciFinder, ChemSpider, and PubMed databases, calculating and predicting using SIRIUS, and processing ion mobility data using MARS.

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