Single cell stable isotope labeled metabolite analysis method based on mass spectrum imaging and application

Abstract

The invention belongs to the technical field of mass spectrometric detection, and particularly relates to a single-cell stable isotope labeled metabolite analysis method based on mass spectrometric imaging and application of the single-cell stable isotope labeled metabolite analysis method. Cells are directly cultured on a conductive glass slide, and stable shaping of single cells is realized by adopting an ammonium acetate cleaning and paraformaldehyde fixing mode. And spraying a substrate on the fixed glass slide attached with the cells, and then carrying out MALDI mass spectrum imaging analysis to obtain the spatial distribution characteristics of the stable isotope labeled metabolites in the single cells. The method can be used for directly and visually analyzing the stable isotope labeled metabolite in a single cell, and has the advantages of simple treatment flow, short analysis time, large detection flux and high detection sensitivity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of mass spectrometry detection, and particularly relates to a single-cell stable isotope labeling metabolite analysis method based on mass spectrometry imaging and application. BACKGROUND

[0002] Cell heterogeneity, as an inherent property of biological systems, is an important driving factor for disease evolution and drug response differences. Carrying out metabolic analysis at the single-cell level is a key prerequisite for analyzing such heterogeneity from a functional perspective. However, traditional metabolomics methods mainly describe the types and contents of metabolites statically, and it is difficult to capture the dynamic changes of cellular metabolic activities. Metabolic flow analysis technology, with the help of stable isotope labeling tracing, can track the flow direction and flow rate of metabolites in the intracellular network in real time, thereby dynamically and functionally revealing the active state of different metabolic pathways under specific physiological or pathological conditions. This method has wide application value in precision medicine, drug target identification, and basic life mechanism research.

[0003] Currently, the analysis method based on liquid chromatography-mass spectrometry (LC-MS) is the mainstream means to realize the detection of stable isotope labeled metabolites in single cells. However, this method inevitably destroys the original spatial structure and microenvironment background of the cells during sample preparation and separation, making it difficult to obtain the original distribution information of metabolites in subcellular regions. In addition, the complex sample pretreatment process is prone to introduce human errors and may cause degradation of some unstable metabolites before analysis. Therefore, it is of great significance to develop a technology that can analyze stable isotope labeled metabolites in single cells in situ and directly. Mass spectrometry imaging technology, as an in situ analysis method without labeling, can directly scan tissue or single-cell samples without complex pretreatment, and simultaneously obtain the molecular composition of hundreds of metabolites and their distribution maps in the original space, providing a new perspective for single-cell metabolic research.

[0004] Although mass spectrometry imaging technology can in situ obtain the molecules and spatial distribution information of hundreds of metabolites at the single-cell level, it still faces a series of key technical difficulties in practical application: the core contradiction is that improving the spatial resolution for achieving single-cell level resolution significantly reduces the detection sensitivity, resulting in weak signals of low-abundance metabolites in cells, which are difficult to accurately quantify. At the same time, single-cell sample preparation is complex and is prone to introduce damage or cause loss of in situ information of metabolites during the processing process; in addition, the technology itself also has the challenges of strong ion suppression effect interference, difficulty in accurate identification of metabolites, and complex analysis of massive spatial data, which jointly restrict the accuracy and wide application of the technology in single-cell metabolic research. SUMMARY

[0005] The application aims to provide a single-cell stable isotope labeled metabolite analysis method based on mass spectrometry imaging and application, so as to overcome the defects of the prior art, directly analyze liver cancer tumor cells, macrophages and fibroblasts by using a matrix assisted laser desorption ionization mass spectrometry imaging technology (MALDI-MSI), and realize the analysis of more than 80 stable isotope labeled metabolites, and the sample pretreatment is simple, the analysis time is short, and the analysis of a large number of samples is facilitated.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the application is: In one aspect, the application provides a single-cell stable isotope labeled metabolite analysis method based on mass spectrometry imaging, comprising the following steps: The single cells are cultured on a conductive slide, and after the stable isotope label is added and cultured, volatile salt solution is used for cleaning and polyformaldehyde fixation treatment. The matrix solution is sprayed on the conductive slide with cells attached, and matrix assisted laser desorption ionization mass spectrometry imaging analysis is performed.

[0007] In some other embodiments, the single cells are one or more of liver cancer tumor cells, macrophages and fibroblasts.

[0008] In some other embodiments, the stable isotope label is D9-choline (D9-PC) and 13 C 18 linoleic acid (LA), arachidonic acid (AA) and 13 C 18- PE34:2.

[0009] In some other embodiments, the conductive slide is an indium tin oxide conductive slide; the surface resistance of the conductive slide is 5-30Ω; preferably, the surface resistance of the conductive slide is 15-25Ω.

[0010] Specifically, the surface resistance of the conductive slide is 15, 20 or 25Ω; more specifically, the surface resistance of the conductive slide is 20Ω. The conductive slide can effectively eliminate the charge accumulation effect on the surface of the sample, and improve the stability, sensitivity and spatial resolution of the analysis results.

[0011] In some other embodiments, the volatile salt solution used is an ammonium acetate solution, and the concentration of the ammonium acetate is 1-10 mol / L; the cleaning method is to place the conductive slide in the ammonium acetate solution for oscillation treatment for multiple cycles, and new ammonium acetate solution is replaced for each cycle, the oscillation rotation speed is 0.5-2 times / s, and the oscillation time is 5-30s.

[0012] Specifically, the concentration of ammonium acetate is 3-7 mol / L; more specifically, the concentration of ammonium acetate is 5 mol / L. Ammonium acetate is volatile by itself and can be completely sublimated and escaped under vacuum or heating conditions, without remaining in the instrument. Using ammonium acetate in this concentration range for cleaning can efficiently remove these interfering salts through ion exchange or dilution displacement, thereby significantly improving the signal-to-noise ratio of the mass spectrum and systematically improving the purity of the sample and the reliability of the mass spectrum analysis. In the method of cleaning, the oscillation speed is 1 time / s, and the oscillation time is 15 s; it has been verified through experiments that by optimizing the control of the ammonium acetate cleaning conditions, the interference of background molecules can be reduced, and the spatial resolution of single-cell mass spectrum imaging is not affected.

[0013] In some other embodiments, the fixing treatment is to place the conductive slide glass in paraformaldehyde and stand for 1-5 min; The concentration of paraformaldehyde is 3-5 wt%.

[0014] Specifically, the fixing time is 1, 2, 3, 4, or 5 min, and the concentration of paraformaldehyde is 3, 4, or 5 wt%. More specifically, the fixing time is 2 min, and the concentration of paraformaldehyde is 4 wt%. Paraformaldehyde can efficiently maintain the morphological structure and antigen epitope of the cell, and maximize the activity and detectability of biomolecules.

[0015] In some other embodiments, the matrix solution is a 1,5-diaminonaphthalene solution; the concentration of the matrix solution is 1-5 mg / mL; preferably, the concentration of the matrix solution is 2-4 mg / mL.

[0016] The solvent of the matrix solution is a mixture of acetonitrile and water, and the mixed volume ratio of acetonitrile and water is (6-8):(2-4).

[0017] Specifically, the concentration of the matrix solution is 2, 3, or 4 mg / mL; and the mixed volume ratio of acetonitrile and water is 6:4, 7:3, or 8:2. More specifically, the concentration of the matrix solution is 3 mg / mL; and the mixed volume ratio of acetonitrile and water is 7:3. 1,5-diaminonaphthalene itself has the property of forming small and uniform crystals, and the use of a lower concentration (1-5 mg / mL) further promotes the formation of ultra-fine nanocrystals. This fine and uniform crystal layer can closely adhere to the cell surface, effectively preventing the lateral diffusion of analytes during matrix covering, thereby preserving the original distribution information of metabolites at the subcellular level. At the same time, the matrix background signal produced by 1,5-diaminonaphthalene itself is very small, so that the signal of low molecular weight metabolites, lipids, and other target molecules will not be masked by strong matrix background peaks, significantly improving the signal-to-noise ratio and analysis sensitivity.

[0018] In some other embodiments, the spraying rate of the matrix solution spraying is 30-100 μL / min, the spraying times are 15-30, the nozzle distance from the sample height is 1-5 mm, and the spraying temperature is 20-60℃; preferably, the nozzle distance from the sample height is 2-4 mm.

[0019] Specifically, the spraying rate of the matrix solution spraying is 75 μL / min, the spraying times are 20, the nozzle distance from the sample height is 3 mm, and the spraying temperature is 40℃.

[0020] In some other embodiments, the single-cell stable isotope-labeled metabolite analysis method further comprises target mass spectrum imaging data extraction of the stable isotope-labeled metabolite, obtaining ion intensity and visualized imaging map thereof. The stable isotope-labeled metabolite comprises one or more of choline, carnitine, betaine, phosphocholine, glycerophosphocholine, phosphatidylcholine, lysophosphatidylcholine, linoleic acid, linolenic acid, arachidonic acid, adrenic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and lysophosphatidylethanolamine.

[0021] In another aspect, the application provides application of the single-cell stable isotope-labeled metabolite analysis method based on mass spectrum imaging in single-cell stable isotope-labeled metabolite analysis.

[0022] The application adopts matrix-assisted laser desorption ionization mass spectrum imaging technology (MALDI-MSI) to directly analyze hepatoma tumor cells, macrophages and fibroblasts, and at least about 1000 cells are needed for one analysis, and the method can be used for analysis of precious samples. More than 80 kinds of stable isotope-labeled metabolites can be analyzed, the sample pretreatment is simple, the analysis time is short, and the analysis of large-scale samples is facilitated.

[0023] The application has the following beneficial effects: (1) The tumor cells, macrophages and fibroblasts are cultured on an indium tin oxide conductive slide, isotopically labeled choline and isotopically labeled linoleic acid are further added in the culture medium, then the cells are washed and fixed, and matrix spraying is performed for mass spectrum imaging visualized analysis. The activity of a specific metabolic pathway is directly associated with the type and spatial position of the cells in a nearly original microenvironment, and the metabolic interaction between cells is intuitively revealed. Stable isotope-labeled metabolites are used to completely eliminate the background interference of complex biological samples, specific and high-sensitivity detection of metabolites is achieved, and metabolic heterogeneity metabolite exchange between cells in different positions of the microenvironment of the same type of cells (such as tumor cells) can be found and explained.

[0024] (2) By washing and fixing cells, the present invention can improve the mass spectrometry imaging detection sensitivity of stable isotope-labeled metabolites, thereby realizing for the first time the visualization characterization of isotope-labeled molecules such as choline, carnitine, betaine, phosphoric acid, glycerophosphocholine, phosphatidylcholine, lysophosphatidylcholine, linoleic acid, linolenic acid, arachidonic acid, adrenal acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and lysophosphatidylethanolamine in liver cancer tumor cells, macrophages, and fibroblasts. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 This is a secondary mass spectrum of choline and stable isotope-labeled D9-choline; Figure 2 It is phosphatidylethanolamine (PE-34:2) and stable isotope labeling. 13 C 18 -Secondary mass spectrum of phosphatidylethanolamine (PE-34:2); Figure 3 This is a mass spectrometry image of stable isotope-labeled metabolites in liver cancer tumor cells in Example 1 of the present invention; Figure 4 This is a mass spectrometry image of stable isotope-labeled metabolites in macrophages in Example 2 of this invention; Figure 5 This is a mass spectrometry image of the stable isotope-labeled metabolites in fibroblasts in Example 3 of the present invention; Figure 6 These are mass spectrometry images of different matrices in Comparative Example 1 of this invention; Figure 7 These are mass spectrometry images corresponding to different nozzle distances from the sample in the spraying method of Comparative Example 2 of this invention; Figure 8 These are mass spectrometry images corresponding to different matrix concentrations in Comparative Example 3 of this invention. Detailed Implementation

[0027] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0028] During the research process, the inventors discovered that sample pretreatment for single-cell mass spectrometry imaging faces more severe challenges compared to biological tissues, specifically as follows: (1) Cell separation and fixation are more complex. Tissue samples can be directly sliced, while single-cell analysis requires obtaining morphologically intact single cells that can be accurately cultured and positioned on conductive glass slides. Metabolites have extremely fast turnover, and the requirements for instantaneous quenching and fixation are much higher than those for tissues. (2) Controlling the uniformity of matrix coating is more difficult. Tissue slices have relatively uniform surfaces, while single cells are tiny, making it extremely difficult to achieve ultra-thin and uniform matrix crystallization on their surfaces. This can easily lead to severe local ion inhibition due to uneven crystallization, causing distortion of metabolite signal responses. These pretreatment deviations, which are negligible at the tissue level, are amplified dramatically at the single-cell scale, becoming the core bottleneck affecting the accuracy of the results.

[0029] Furthermore, current single-cell stable isotope-labeled metabolite analysis techniques can disrupt the original spatial morphology and microenvironment information of cells. In addition, the pretreatment process may introduce biases or lead to the degradation of some unstable metabolites in cells, which seriously affects the accurate analysis of stable isotope-labeled metabolites in single cells.

[0030] In view of this, the present invention provides a method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging technology, the method comprising: (1) Different types of cells were cultured into 24-well culture plates, and a circular indium tin oxide conductive glass slide was placed at the bottom of the culture plate.

[0031] (2) After adding stable isotope-labeled metabolites to the culture system in step (1), culture for 48 hours; (3) Place the conductive glass slide with cells attached obtained in step (2) into a staining jar containing a volatile salt solution; (4) Place the dyeing vat from step (3) on a shaker and vibrate it; (5) Place the conductive slide obtained in step (4) into a staining tank containing fixative for cell fixation; (6) Place the conductive glass slide treated in step (5) in a vacuum desiccator and dry for 10 min; (7) Prepare the matrix solution; (8) Spray the conductive glass slide processed in step (6) with a 1,5-diaminonaphthalene matrix solution. (9) Perform MALDI mass spectrometry imaging analysis on the conductive glass slide after the matrix has been sprayed in step (8); (10) Targeted mass spectrometry imaging data of isotope-labeled metabolite molecules in cells are extracted to obtain their ion intensity and visualization images.

[0032] The minimum culture density of this invention is 1000 cells / well. If the density is too low, the number of single cells attached to the conductive slide will be too small, making effective detection impossible. If the density is too high, the cells will aggregate, affecting the spatial resolution of the detection and making it impossible to achieve accurate characterization at the single-cell level.

[0033] In mass spectrometry imaging analysis, the type of matrix, spraying method, and concentration all affect the sensitivity and spatial resolution of metabolite analysis. This invention, through continuous screening and optimization, finally determined the above-mentioned matrix type and matrix spraying conditions, thereby improving the detection sensitivity of metabolites while ensuring spatial resolution, and realizing in-situ visualization detection of isotope-labeled metabolites at the single-cell level.

[0034] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0035] Example 1 Mass spectrometry imaging analysis of stable isotope-labeled metabolites in liver cancer tumor cells includes the following steps: (1) Hep 3B liver cancer cells were cultured in a 24-well culture plate with a circular indium tin oxide conductive glass slide placed at the bottom.

[0036] (2) Add 100 μmol / L of D9-choline and... to the petri dish respectively. 13 C 18 - Linoleic acid, cultured for 48 hours.

[0037] (3) Prepare a 5 mol / L ammonium acetate solution, place the conductive glass slide in the ammonium acetate solution and place it on a shaker, shake at a shaking speed of 1 oscillation / s for 15 s, replace with a new 5 mol / L ammonium acetate solution and shake again at a shaking speed of 1 oscillation / s for 15 s, repeat three times.

[0038] (4) Place the cleaned conductive slide with attached cells in 4% paraformaldehyde for 2 min to fix it.

[0039] (5) Place the fixed conductive slide with attached cells in a vacuum dryer and dry for 10 min.

[0040] (6) Weigh 150 mg of 1,5-diaminonaphthalene reagent and place it in a 50 mL reagent bottle. Add 50 mL of acetonitrile:water (70:30, v / v) solution, vortex to mix, and sonicate for 5 min to obtain a 3.0 mg / mL 1,5-diaminonaphthalene solution.

[0041] (7) A 3.0 mg / mL solution of 1,5-diaminonaphthalene was sprayed onto the surface of a conductive glass slide with attached cells using a Matrix Builder matrix sprayer. The spraying rate was 75 μL / min, the number of spraying cycles was 20, the nozzle distance from the sample was 3 mm, and the spraying temperature was 40℃.

[0042] (8) The conductive glass slides after the matrix was sprayed were analyzed by full scan at m / z 100~1000 using a Bruker Rapiflex MALDI-TOF / TOF mass spectrometer to target and extract stable isotope-labeled metabolite ions. The detected stable isotope-labeled metabolites are shown in Table 1.

[0043] (9) Visual imaging analysis of stable isotope-labeled metabolite ions in liver cancer tumor cells was performed using SCiLS Lab 2018b software. Representative isotope-labeled metabolites are shown in the figure. Figure 3 .

[0044] Table 1. Stable isotope-labeled metabolites detected in hepatocellular carcinoma tumor cells.

[0045] Table 1 shows that this method can detect 82 stable isotope-labeled metabolites in tumor cells. Figure 1 It can be seen that secondary mass spectrometry analysis can determine the presence of stable isotope-labeled choline in single cells.

[0046] Depend on Figure 2 It can be seen that the presence of stable isotope-labeled phosphatidylethanolamine in single cells can be confirmed by secondary mass spectrometry analysis.

[0047] Depend on Figure 3 It can be seen that this method can achieve mass spectrometry imaging analysis of isotope-labeled metabolites in liver cancer tumor cells.

[0048] Example 2 Mass spectrometry imaging analysis of stable isotope-labeled metabolites in macrophages includes the following steps: (1) M0 macrophages were cultured in 24-well culture plates with a circular indium tin oxide conductive glass slide placed at the bottom.

[0049] (2) Add 100 μmol / L of D9-choline and... to the petri dish respectively. 13 C 18 - Linoleic acid, cultured for 48 hours.

[0050] (3) Prepare a 5 mol / L ammonium acetate solution, place the conductive glass slide in the ammonium acetate solution and place it on a shaker, shake at a shaking speed of 1 oscillation / s for 15 s, replace with a new 5 mol / L ammonium acetate solution and shake again at a shaking speed of 1 oscillation / s for 15 s, repeat three times.

[0051] (4) Place the cleaned conductive slide with attached cells in 4% paraformaldehyde for 2 min to fix it.

[0052] (5) Place the fixed conductive slide with attached cells in a vacuum dryer and dry for 10 min.

[0053] (6) Weigh 150 mg of 1,5-diaminonaphthalene reagent and place it in a 50 mL reagent bottle. Add 50 mL of acetonitrile:water (70:30, v / v) solution, vortex to mix, and sonicate for 5 min to prepare a 3.0 mg / mL 1,5-diaminonaphthalene solution.

[0054] (7) A 3.0 mg / mL solution of 1,5-diaminonaphthalene was sprayed onto the surface of a conductive glass slide with attached cells using a Matrix Builder matrix sprayer. The spraying rate was 75 μL / min, the number of spraying cycles was 20, the nozzle distance from the sample was 3 mm, and the spraying temperature was 40℃.

[0055] (8) The conductive glass slides after the matrix was sprayed were analyzed by full scan at m / z 100~1000 using a Bruker Rapiflex MALDI-TOF / TOF mass spectrometer to target and extract stable isotope-labeled metabolite ions. The detected stable isotope-labeled metabolites are shown in Table 2.

[0056] (9) Visual imaging analysis of stable isotope-labeled metabolite ions in macrophages was performed using SCiLS Lab 2018b software. Representative isotope-labeled metabolites are shown in the figure. Figure 4 .

[0057] Table 2 Stable isotope-labeled metabolites detected in macrophages

[0058] Table 2 shows that this method can detect 78 stable isotope-labeled metabolites in macrophages. Figure 4 It can be seen that this method can achieve mass spectrometry imaging analysis of isotope-labeled metabolites in macrophages.

[0059] Example 3 Mass spectrometry imaging analysis of stable isotope-labeled metabolites in fibroblasts includes the following steps: (1) Fibroblasts were cultured in 24-well culture plates with a circular indium tin oxide conductive glass slide placed at the bottom.

[0060] (2) Add 100 μmol / L of D9-choline and... to the petri dish respectively. 13 C 18 - Linoleic acid, cultured for 48 hours.

[0061] (3) Prepare a 5 mol / L ammonium acetate solution, place the conductive glass slide in the ammonium acetate solution and place it on a shaker, shake at a shaking speed of 1 oscillation / s for 15 s, replace with a new 5 mol / L ammonium acetate solution and shake again at a shaking speed of 1 oscillation / s for 15 s, repeat three times.

[0062] (4) Place the cleaned conductive slide with attached cells in 4% paraformaldehyde for 2 min to fix it.

[0063] (5) Place the fixed conductive slide with attached cells in a vacuum dryer and dry for 10 min.

[0064] (6) Weigh 150 mg of 1,5-diaminonaphthalene reagent and place it in a 50 mL reagent bottle. Add 50 mL of acetonitrile:water (70:30, v / v) solution, vortex to mix, and sonicate for 5 min to prepare a 3.0 mg / mL 1,5-diaminonaphthalene solution.

[0065] (7) A 3.0 mg / mL solution of 1,5-diaminonaphthalene was sprayed onto the surface of a conductive glass slide with attached cells using a Matrix Builder matrix sprayer. The spraying rate was 75 μL / min, the number of spraying cycles was 20, the nozzle distance from the sample was 3 mm, and the spraying temperature was 40℃.

[0066] (8) The conductive glass slides after the matrix was sprayed were analyzed by full scan at m / z 100~1000 using a Bruker Rapiflex MALDI-TOF / TOF mass spectrometer to target and extract stable isotope-labeled metabolite ions. The detected stable isotope-labeled metabolites are shown in Table 3.

[0067] (9) Visual imaging analysis of stable isotope-labeled metabolite ions in fibroblasts was performed using SCiLS Lab 2018b software. Representative isotope-labeled metabolites are shown in the figure. Figure 5 .

[0068] Table 3 lists the stable isotope-labeled metabolites detected in fibroblasts.

[0069] Table 3 shows that this method can detect 78 stable isotope-labeled metabolites in fibroblasts. Figure 5 It can be seen that this method can achieve mass spectrometry imaging analysis of isotope-labeled metabolites in fibroblasts.

[0070] Comparative Example 1 Unlike Example 1, the matrix was optimized, while the other steps were the same as in Example 1. Specifically, 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and 1,5-diaminonaphthalene (1,5-DAN) were used as matrices, and the results are as follows. Figure 6 As shown.

[0071] like Figure 6 The study found that 1,5-diaminonaphthalene (1,5-DAN) showed the best results. This may be because 1,5-DAN has a unique charge-transfer ionization mechanism, which can more efficiently ionize metabolites lacking easily protonated sites to form stable molecular ion peaks. At the same time, its mild ionization process can effectively suppress fragmentation, thereby obtaining a more sensitive mass spectrometry signal.

[0072] Comparative Example 2 Unlike Example 1, the nozzle-to-sample height in the spraying method was optimized; other steps remained the same as in Example 1. Specifically, the nozzle-to-sample heights were 1 mm, 3 mm, and 5 mm. The results are as follows: Figure 7 As shown.

[0073] like Figure 7 The study found that the optimal effect was achieved when the nozzle was 3 mm above the sample. This is likely because if the nozzle is too low, the matrix solution will not be sufficiently atomized, while if the nozzle is too high, the matrix will not co-crystallize well with the metabolites on the sample surface.

[0074] Comparative Example 3 Unlike Example 1, the matrix concentration was optimized, while the other steps were the same as in Example 1. Specifically, the matrix concentrations were 1 mg / mL, 3 mg / mL, and 5 mg / mL. The results are as follows: Figure 8 As shown.

[0075] like Figure 8 The study found that the detection sensitivity of metabolites was insufficient at 1 mg / mL, and the spatial resolution of metabolite detection was affected at 5 mg / mL. Therefore, a 1,5-DAN matrix concentration of 3 mg / mL was chosen. This may be because when the matrix concentration is too low, it cannot effectively assist in the ionization of metabolites, while too high a concentration may lead to a significant increase in the matrix effect, affecting the detection of metabolites.

[0076] In summary, the type, spraying method, and concentration of the matrix all affect the sensitivity and spatial resolution of metabolite analysis during mass spectrometry imaging analysis. Through continuous screening and optimization, this invention has finally determined the above-mentioned matrix type and matrix spraying conditions, thereby improving the detection sensitivity of metabolites while ensuring spatial resolution, and realizing in-situ visualization detection of isotope-labeled metabolites at the single-cell level.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging, characterized in that, Includes the following steps: Single cells were cultured on conductive glass slides, and stable isotope markers were added for further culture. After culture, the cells were washed with volatile salt solution and fixed with paraformaldehyde. The matrix solution was sprayed onto a conductive glass slide with attached cells for matrix-assisted laser desorption / ionization mass spectrometry imaging analysis.

2. The method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging according to claim 1, characterized in that, The single cell is one or more of liver cancer tumor cells, macrophages, and fibroblasts.

3. The method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging according to claim 1, characterized in that, The stable isotope markers are D9-choline and 13 C 18 - Linoleic acid.

4. The method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging according to claim 1, characterized in that, The conductive glass slide is an indium tin oxide conductive glass slide; the surface resistance of the conductive glass slide is 5-30Ω.

5. The method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging according to claim 1, characterized in that, The volatile salt solution is an ammonium acetate solution, and the concentration of the ammonium acetate solution is 1-10 mol / L; The cleaning method involves placing a conductive glass slide in an ammonium acetate solution and oscillating it for multiple cycles, replacing the ammonium acetate solution with a fresh solution for each cycle. The oscillation speed is 0.5-2 times / s, and the oscillation time is 5-30s.

6. The method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging according to claim 1, characterized in that, The fixation process involves placing the conductive glass slide in paraformaldehyde and allowing it to stand for 1-5 minutes to fix it. The concentration of paraformaldehyde is 3-5 wt%.

7. The method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging according to claim 1, characterized in that, The matrix solution is a 1,5-diaminonaphthalene solution; the concentration of the matrix solution is 1-5 mg / mL; The solvent of the matrix solution is a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water is (6-8):(2-4).

8. The method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging according to claim 1, characterized in that, The spraying rate of the matrix solution is 30~100 μL / min, the number of sprays is 15~30, the nozzle distance from the sample is 1~5 mm, and the spraying temperature is 20~60℃. Preferably, the matrix solution is sprayed at a rate of 75 μL / min, with 20 sprays, a nozzle distance of 3 mm from the sample, and a spraying temperature of 40°C.

9. The method for analyzing stable isotope-labeled metabolites in a single cell based on mass spectrometry imaging according to claim 1, characterized in that, The single-cell stable isotope-labeled metabolite analysis method also includes extracting targeted mass spectrometry imaging data from the stable isotope-labeled metabolites to obtain their ion intensity and visualization imaging map. The stable isotope-labeled metabolites include one or more of choline, carnitine, betaine, phosphoric acid choline, glycerophosphocholine, phosphatidylcholine, lysophosphatidylcholine, linoleic acid, linolenic acid, arachidonic acid, adrenal acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and lysophosphatidylethanolamine.

10. The application of the single-cell stable isotope-labeled metabolite analysis method based on mass spectrometry imaging as described in any one of claims 1-9 in the analysis of single-cell stable isotope-labeled metabolites.

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