Disease single cell high-throughput fine lipidomics analysis method and application thereof

Abstract

The invention discloses a disease single cell high-throughput fine lipidomics analysis method and application thereof. The invention discloses a single cell fine lipidomics high-throughput analysis method based on combination of ozone induced cracking and electrospray ionization mass spectrometry flow type combination. The method is simple, complex cell sample pretreatment is not needed, basic lipidomics information and fine lipidomics information of single cells can be synchronously obtained in real time in a high-throughput mode, and lipid compounds in the single cells are accurately analyzed and identified; the method is of great significance in disclosing a lipid metabolism regulation mechanism and researching disease targets at a single cell level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a disease single-cell high-throughput fine lipidomics analysis method and application thereof. BACKGROUND

[0002] Lipids are the main chemical substances of cells in living organisms, and play an important role in key physiological processes such as dynamic assembly of cell membranes, regulation of cell energy homeostasis and transmembrane signal transduction. The lipid composition of cells is highly complex and dynamic, and a large number of lipid isomers are formed by different fatty chain lengths and configurations, different unsaturation degrees, and different carbon-carbon double bond (C=C) positions. The subtle changes in the structure of lipid molecules, especially the number, position and configuration of unsaturated C=C in the fatty chain, can all lead to changes in the cell microenvironment. The occurrence and development of various diseases such as atherosclerosis, Alzheimer's disease and cancer are closely related to abnormal lipid metabolism in cells.

[0003] Traditional lipidomics analysis methods are based on the average data analysis of a large number of cell populations, and cannot reveal the differences in lipid metabolism between different cell individuals, nor can they reflect the impact of abnormal lipid metabolism in individual cells on the overall function of the organism. Studies have shown that even cell populations with the same genotype (such as cancer cells in the tumor microenvironment), their lipid composition also has significant heterogeneity, which directly affects the functional state and treatment response of cells. Currently, the research of life sciences and analytical chemistry is gradually entering the microscopic level, and single-cell lipidomics research can overcome the masking of individual information by population analysis averages, thereby more accurately revealing the relevance between abnormal lipid metabolism in cells and the molecular mechanisms of diseases. However, high-throughput, high-sensitivity, high-coverage single-cell lipidomics analysis methods still face major technical challenges.

[0004] Mass spectrometry has unique advantages in single-cell analysis. A series of single-cell mass spectrometry analysis techniques such as single-probe MS, pico-ESI-MS and inESI-MS have emerged in recent years, which to some extent realize the lipidomics analysis research at the single-cell level. However, these methods are complex and difficult to operate, and have low detection throughput, making it difficult to achieve high-throughput lipidomics analysis of large-scale single-cell samples.

[0005] Mass cytometry is a technique for high-throughput analysis of single cells. Classical mass cytometry uses inductively coupled plasma mass spectrometry (ICP-MS) as the detection unit and requires labeling with metal probes. In recent years, label-free single-cell organic mass cytometry has rapidly developed. High-throughput single-cell analysis techniques such as electrospray ionization mass cytometry (CyESI-MS), intact live-cell electroemission ionization mass spectrometry (ILCEI-MS), and plasma-assisted electrospray ionization mass cytometry (PACyESI-MS) have successfully detected hundreds of metabolites in single cells. The Deep Organic Mass Cytometry Single-Cell Metabolomics Platform (ID-organic cytoMS) extends single-cell analysis time while maintaining analytical throughput through efficient online cell lysis and non-contact ESI-MS technology, enabling the acquisition of multi-stage mass spectrometry data for metabolite identification and isomer differentiation. Combining the Pasternò-Büchi photochemical reaction with CyESI-MS allows for high-throughput single-cell lipidomics analysis while also providing information on the C=C positions and isomers of intracellular unsaturated lipids. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of existing single-cell lipidomics analysis techniques and provide a high-throughput, refined single-cell lipidomics analysis method that can acquire basic and refined lipidomics information of a single cell in a high-throughput, synchronous, and real-time manner.

[0007] Another objective of this invention is to provide the application of the above-described high-throughput, refined lipidomics analysis method for single-cell analysis of diseases.

[0008] The objective of this invention is achieved through the following technical solution: A high-throughput, refined lipidomics analysis method for single-cell disease analysis includes the following steps: (1) Prepare cell sample suspensions using methanol-ammonium formate mixed solution, analyze cell suspensions using electrospray mass cytometry, record analysis results, and obtain basic lipidomics information of single cells; (2) O3 was introduced into the suspension prepared in step (1) to carry out an ozone-induced dissociation (OzID) reaction. By precisely controlling the conditions of the OzID reaction, ~50% of the unsaturated lipids C=C in the cell suspension underwent the OzID reaction to produce characteristic fragments, while the lipids that did not undergo the OzID reaction retained their original structure. After the reaction, electrospray mass cytometry was used for analysis, and the analysis results were recorded to obtain refined lipidomics information for single cells. (3) By comparing the analysis results obtained in steps (1) and (2), the statistical analysis and identification of single-cell basic lipid fingerprint and fine lipid fingerprint can be used to achieve the purpose of distinguishing disease cell types and subtypes, studying the regulatory mechanism of disease lipid metabolism and disease targets.

[0009] The cell sample to be tested in step (1) is at least one of BEAS-2B cells, A549 cells, H1299 cells, HL-60 cells, and Jurkat cells.

[0010] The methanol-ammonium formate mixed solution mentioned in step (1) is prepared by adding methanol to an aqueous solution of ammonium formate, and the concentration of ammonium formate is 100-200 mM.

[0011] The volume fraction of methanol in the methanol-ammonium formate mixed solution in step (1) is 0-40%; preferably 20%.

[0012] The concentration of the cell sample suspension in step (1) is 3–8 × 10⁻⁶. 4 cells / mL.

[0013] The specific steps of the electrospray mass cytometry described in steps (1) and (2) are as follows: Cell suspension was flowed at a rate of 1 μL / min through a 25 μm inner diameter capillary via a precision flow rate or pressure controlled pump. A 1.5 kV DC voltage was applied at the conductive interface connecting the capillary and the pump to create a stable electrospray ionization for mass spectrometry detection. The mass spectrometry was performed in full scan mode, with a scanning mass range of... m / z =100~1000, quality resolution 70000.

[0014] The conditions for the OzID reaction in step (2) are an O3 purge flow rate of 0.1 to 0.5 L / min and an O3 purge reaction time of 10 to 30 s; preferably, the O3 flow rate is 0.1 L / min and the reaction time is 20 s.

[0015] The above-mentioned single-cell high-throughput fine lipidomics analysis method for diseases is applied in the identification of different types of disease cells and subtypes.

[0016] The application of the high-throughput fine lipidomics analysis method for single-cell disease analysis in identifying different types of disease cells and subtypes includes the following steps: performing high-throughput fine lipidomics analysis on single-cell disease cells, recording fine lipid fingerprints of different types of disease cells and subtypes, distinguishing different types of disease cells and subtypes through statistical analysis, and obtaining lipid marker information of different types of disease cells and subtypes through mass spectrometry identification.

[0017] The above-mentioned high-throughput fine lipidomics analysis method for single cells was applied to the analysis of the proportion of different lipid isoforms in single cells.

[0018] A high-throughput single-cell identification method for a disease includes the following steps: The aforementioned high-throughput, refined lipidomics analysis method for single cells was used to detect known cell lines and record characteristic fingerprint profiles. Then, the cells to be tested were analyzed and compared with the characteristic fingerprint profiles to obtain identification results.

[0019] A single-cell high-throughput identification system for diseases includes detection equipment and reagents for single-cell high-throughput identification methods for non-disease diagnosis and treatment purposes, as well as a data processing module for analysis results.

[0020] The single cells mentioned include at least one of BEAS-2B cells, A549 cells, H1299 cells, HL-60 cells, and Jurkat cells.

[0021] The present invention has the following advantages and effects compared with the prior art: (1) OzID reaction was performed on unsaturated lipids in the single cells to be analyzed using O3. By precisely controlling the OzID reaction conditions, approximately 50% of the C=C unsaturated lipids in the cells underwent the OzID reaction to produce characteristic fragments, while the lipids that did not undergo the OzID reaction retained their original structure. After the OzID reaction, C=C produced two characteristic ion fragments with a mass number difference of 48 Da. Among them, the aldehyde ion fragment with the smaller mass number was the main characteristic fragment ion, while the methoxy-hydroxy adduct ion with a larger mass of 48 Da served as an auxiliary diagnostic characteristic fragment ion. Therefore, without the need for multi-stage mass spectrometry analysis, high-throughput and simultaneous acquisition of lipid compound information and unsaturated lipid C=C positional isomer information in single cells was achieved.

[0022] (2) The method of the present invention is simple and does not require labeling or complex cell sample pretreatment. It can obtain basic and fine lipidomics information of a single cell in a high-throughput, synchronous and real-time manner.

[0023] (3) This invention develops a high-throughput single-cell fine lipidomics analysis method based on the combination of OzID reaction and electrospray mass cytometry. The method is of great significance for revealing the lipid metabolism regulation mechanism and disease targets at the single-cell level. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the process of using OzID reaction coupled with electrospray mass cytometry for high-throughput, fine-grained lipidomics analysis of single cells in diseases.

[0025] Figure 2This is the electrospray mass spectrometry flow cytometry analysis mass spectrum of phosphatidylcholine PC 16:0 / 18:1(Δ9) in Example 1; where A to F are the results of methanol ratios of 0%, 5%, 10%, 20%, 30% and 40%, respectively, and G is the mechanism of OzID in PC 16:0 / 18:1(Δ9).

[0026] Figure 3 The mass spectra of lung cells obtained by BEAS-2B electrospray mass cytometry analysis in Example 2 are shown; where A to D are the results of methanol proportions of 10%, 20%, 30%, and 40%, respectively.

[0027] Figure 4 The mass spectra obtained from the BEAS-2B electrospray mass cytometry analysis of lung cells in Example 2 are shown; where A to F are the results of OzID reaction times of 10 s, 20 s, 30 s, 40 s, 50 s, and 60 s, respectively.

[0028] Figure 5 This is a pulse chromatogram obtained from the BEAS-2B electrospray mass cytometry analysis of lung cells in Example 2; where A to D represent ion... m / z 760.5851 (PC(16:0 / 18:1)), m / z 678.4704 (PC(16:0 / 18:1(Δ8))), m / z 650.4391 (PC(16:0 / 18:1(Δ9))) and m / z The result of 636.42356 (PC(16:0 / 18:1(Δ11))).

[0029] Figure 6 These are mass spectra obtained from electrospray mass cytometry analysis of different cell types in Example 3; where A is the mass spectra obtained from electrospray mass cytometry analysis of lung cells BEAS-2B, B is the mass spectra of lung cells BEAS-2B after OzID reaction, C is the mass spectra of lung cancer cells A549, D is the mass spectra of lung cancer cells A549 after OzID reaction, E is the mass spectra of lung cancer cells H1299 after OzID reaction, F is the mass spectra obtained from electrospray mass cytometry analysis of lung cancer cells H1299 after OzID reaction, G is the mass spectra of different cell types and the differences in intercellular lipid characteristics shown by UMAP analysis after OzID reaction, and H is the mass spectra of diagnostic ions of different cell types after OzID reaction (…). m / z 566.3089 and m / z A comparison chart of expression levels (678.4704).

[0030] Figure 7This is the main lipid isoform data identified in BEAS-2B, A549, and H1299 cells using OzID electrospray mass cytometry in Example 3.

[0031] Figure 8 This is the main lipid isoform data identified in BEAS-2B, A549, and H1299 cells using OzID electrospray mass cytometry in Example 3.

[0032] Figure 9 This is the main lipid isoform data identified in BEAS-2B, A549, and H1299 cells using OzID electrospray mass cytometry in Example 3.

[0033] Figure 10 This is the main lipid isoform data identified in BEAS-2B, A549, and H1299 cells using OzID electrospray mass cytometry in Example 3.

[0034] Figure 11 These are mass spectra obtained from electrospray mass cytometry analysis of different cell types in Example 4; where A represents HL-60 leukemia cells, B represents HL-60 leukemia cells after OzID reaction, C represents Jurkat leukemia cells, D represents Jurkat leukemia cells after OzID reaction, E represents the differences in intercellular lipid characteristics shown by UMAP analysis after OzID reaction for different cell types, and F represents the diagnostic ions (DIE) of HL-60 and Jurkat cells after OzID reaction. m / z 636.4235 and m / z A comparison chart of expression levels (690.4704). Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0036] Example 1: Optimization of OzID reaction conditions for PC 16:0 / 18:1 (Δ9) standard solution 1.1 Preparation of methanol-ammonium formate mixed solution An aqueous solution with a concentration of 140 mM was prepared using ammonium formate. Then, 0%, 5%, 10%, 20%, 30%, and 40% (volume fraction) of methanol were added to prepare methanol-ammonium formate mixed solutions with different proportions of methanol.

[0037] 1.2 Preparation of PC 16:0 / 18:1 (Δ9) standard solution Take the methanol-ammonium formate mixed solution prepared in 1.1, add PC 16:0 / 18:1(Δ9), stir well to obtain a PC 16:0 / 18:1(Δ9) standard solution with a concentration of 1 mg / mL.

[0038] 1.3 OzID reaction and mass spectrometry detection The PC 16:0 / 18:1 (Δ9) standard solution prepared in step 1.2 was passed into O3 at a flow rate of 0.1 L / min and reacted for 20 s. The solution was then analyzed by electrospray ionization mass cytometry. The specific steps are as follows: A PC 16:0 / 18:1(Δ9) standard solution was flowed at a rate of 1 μL / min through a 25 μm inner diameter capillary via a precision flow rate controlled pump. A 1.5 kV DC voltage was applied at the conductive interface connected to the capillary to form a stable electrospray ionization for mass spectrometry detection. The mass spectrometer was used in full scan mode. m / z (100-1000, quality resolution 70,000), simultaneously detecting lipid molecules and OzID fragments.

[0039] 1.4 Experimental Results and Discussion Experimental results are as follows Figure 2 As shown, in a pure ammonium formate aqueous solution, PC 16:0 / 18:1 (Δ9) mainly produces m / z The protonated ion at 760.5851 has an extremely weak OzID fragmentation signal. Figure 2 (A) This is because the reaction of O3 oxygen with the Δ9 double bond first generates a primary ozonide ([1,2,3]-trioxacyclopentane), which rapidly rearranges to the [1,2,4]-trioxacyclopentane intermediate. This intermediate reacts with water in aqueous solution to form an unstable intermediate. α -Hydroxyhydroperoxides eventually decompose into small molecules with no diagnostic value.

[0040] However, as the proportion of methanol increases, the OzID efficiency improves significantly (5%). Figure 2 B; 10%, Figure 2 C; 20%, Figure 2 D; 30%, Figure 2 China E; 40%, Figure 2 (F). Methanol reacts with the intermediate under neutral conditions at a rate more than 100 times faster than water, competitively capturing the [1,2,4]-trioxacyclopentane intermediate to generate a stable intermediate. α -Methoxyhydroperoxide. This product further cleaves to produce two characteristic diagnostic ions: C9-C... 10 Aldehyde fragment ions formed by bond breaking m / z 650.4391, and fragment ions of methoxy-hydroxy adducts formed by methanol addition. m / z 698.4604, the mass number difference between the two characteristic ion fragments is 48 Da ( Figure 2 (G). Among them, m / z 650.4394 aldehyde group fragments are the main characteristic fragment ions. m / z The 698.4604 methoxy-hydroxy adduct ion level serves as a characteristic fragment ion for auxiliary diagnosis. The abundance of these fragment ions increases significantly with increasing methanol concentration—reaching the highest abundance at 40% methanol concentration, which is almost undetectable in pure aqueous solution. This demonstrates that methanol functions as both a nucleophilic trap and a stabilizer: the electronic effect of the methoxy group prolongs the intermediate lifetime, allowing previously transient intermediates to generate fragments with structural diagnostic significance. This establishes the indispensability of methanol for achieving efficient OzID reactions in this analytical system.

[0041] Example 2: Optimization of OzID reaction conditions for BEAS-2B lung cells 2.1 Cell Culture and Preparation of Cell Suspension BEAS-2B lung cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C in a humidified incubator with 5% CO2, and passaged every 2-3 days. When the cell density reached 80%-90%, the cells were collected by centrifugation (1000 rpm, 3 min) and washed twice with phosphate-buffered saline (PBS). After centrifugation again (1000 rpm, 3 min) to remove the PBS, the cells were resuspended in methanol-ammonium formate solutions prepared in Example 1 at different ratios (10%, 20%, 30%, 40%), and the cell density was controlled to be approximately 5 × 10⁶ cells / year by cell counting. 4 cells / mL.

[0042] 2.2 Determination of the reaction efficiency of OzID with methanol-ammonium formate mixed solutions of different proportions Take an appropriate amount of the lung cell suspension prepared in 2.1, introduce O3 and perform OzID reaction for 20 s, and then perform high-throughput single-cell mass cytometry analysis using electrospray mass cytometry. The mass spectrometry conditions are as described in 1.3 of Example 1, and the cell suspension flow rate is set to 1 μL / min. Record the positive ion mass spectrum and obtain the mass spectrometric fingerprint of BEAS-2B cells after the OzID reaction through high-throughput single-cell analysis.

[0043] The results are as follows Figure 3As shown, experimental data indicate that when the methanol content is 10%, the characteristic ions produced by the OzID reaction... m / z The relative proportion of 650.4391 is relatively low. Figure 3 (A) This demonstrates that the OzID reaction efficiency is low; when the methanol ratio is 20%, the characteristic ions produced by the reaction... m / z 650.4391 and cell characteristic ions m / z Both 760 and 5851 are relatively high. Figure 3 (B) This demonstrates that the OzID reaction efficiency is good; further increasing the methanol content (30%) can improve the OzID efficiency, but... m / z The intensity of characteristic lipid ions in cells 760.5851 was significantly reduced. Figure 3 (C) When the methanol ratio reaches 40%, m / z The signal of characteristic lipid ions in 760.5851 cells almost disappeared. Figure 3 (D). The optimal solution was finally determined to be a methanol-ammonium formate mixed solution containing 20% ​​methanol. This formulation can ensure sufficient OzID reaction efficiency, maintain good cell integrity, and obtain clear fragment spectra of endogenous lipids and OzID reaction characteristics.

[0044] 2.3 Detection of the effect of different O3 purging reaction times on OzID reaction efficiency The experiment was conducted according to the method in 2.2. The cell suspension used was a methanol-ammonium formate mixed solution containing 20% ​​methanol. The O3 purge time for the OzID reaction was set to 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, and 50 seconds, respectively. Then, high-throughput single-cell analysis was performed by electrospray mass cytometry with the cell suspension flow rate set to 1 μL / min. Positive ion mass spectra were recorded to obtain the mass spectrometric fingerprints of BEAS-2B lung cells after different OzID reaction times.

[0045] Experimental results are as follows Figure 4 As shown in the mass spectrum obtained from the experiment, the reaction time was 5 seconds, and only a small portion of the C=C of the unsaturated lipids underwent the OzID reaction. Figure 4 (A); the reaction time is 10 seconds, and the proportion of C=C undergoing the OzID reaction increases significantly ( Figure 4 (B); the reaction time is 20 seconds, and the proportion of C=C undergoing the OzID reaction is approximately 50%, at which point the lipid fingerprint information is most abundant. Figure 4 (C); Continue to increase the O3 purging reaction time, m / zThe peaks of major lipid compounds in cells, such as 760 and 5851, were significantly reduced (30 seconds). Figure 4 Middle D, 40 seconds Figure 4 (E); when the O3 purging reaction time is 50 seconds, m / z The signal of characteristic lipid ions in 760.5851 cells almost disappeared. Figure 4 (Middle F).

[0046] To obtain the most information-rich fingerprint spectrum, the purge reaction time with O3 at approximately 50% OzID reaction efficiency should be optimized to achieve the most detailed single-cell lipid fingerprint spectrum. The results demonstrate that for single-cell detection, a purge reaction time of 20 seconds with O3 is optimal. Under this optimized condition, both endogenous lipids and OzID reaction products in the cells obtain synchronous and ideal pulse signals. Figure 5 ).

[0047] Example 3: Fine lipid fingerprinting and differentiation of BEAS-2B lung cells and A549 and H1299 lung cancer cells 3.1 Cell Culture Lung cells BEAS-2B, lung cancer cells A549, and lung cancer cells H1299 were placed in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, respectively, and cultured in a humidified incubator at 37°C and 5% CO2, passaged every 2-3 days. When the cell density reached 80%-90%, the cells were collected by centrifugation (1000 rpm, 3 min) and washed twice with phosphate-buffered saline (PBS). After centrifugation again (1000 rpm, 3 min) to remove the PBS, the cells were resuspended in 140 mM ammonium formate aqueous solution containing 20% ​​methanol (pH=7.3), and the cell density was controlled to be approximately 5 × 10⁶ cells / year by cell counting. 4 Cell suspensions of lung cells BEAS-2B, lung cancer cells A549, and lung cancer cells H1299 were obtained by dividing the cells / mL.

[0048] 3.2 Obtaining mass spectrometric fingerprints using high-throughput single-cell mass cytometry analysis The cell suspensions of lung cells BEAS-2B, lung cancer cells A549, and lung cancer cells H1299 prepared in step 3.1 were analyzed by high-throughput single-cell mass cytometry. The cell suspension flow rate was set to 1 μL / min, and positive ion mass spectra were recorded to obtain the basic mass spectrometric fingerprints of lung cells BEAS-2B, lung cancer cells A549, and lung cancer cells H1299. Subsequently, O3 was introduced to perform an OzID reaction on lung cells BEAS-2B, lung cancer cells A549, and lung cancer cells H1299, with an O3 flow rate of 0.1 L / min and a reaction time of 20 seconds. Then, high-throughput single-cell analysis was performed by electrospray mass cytometry, with the cell suspension flow rate set to 1 μL / min, and positive ion mass spectra were recorded to obtain the fine mass spectrometric fingerprints of lung cells BEAS-2B, lung cancer cells A549, and lung cancer cells H1299 after the OzID reaction.

[0049] 3.3 Experimental Results Experimental results are as follows Figures 6-10 As shown, where Figure 6 Images A through E show the mass spectrometric fingerprints of lung cells BEAS-2B, lung cancer cells A549, and lung cancer cells H1299 before and after the OzID reaction. Electrospray ionization mass cytometry analysis of BEAS-2B, A549, and H1299 cells, without the OzID reaction, detected a total of 21 major endogenous lipid compound signals. These signals mainly include: m / z 703.5748, 732.5538, 760.5851, 782.5694, 786.6007, and 808.5851, etc. Figure 6 A, C, E Figure 7 , Figure 8 , Figure 9 , Figure 10 These lipid compounds were further analyzed by UHPLC-MS and UHPLC-MS / MS, revealing a total of 29 isomers. Figure 7 , Figure 8 , Figure 9 , Figure 10 To obtain refined lipidomics information at the single-cell level, OzID coupled with electrospray ionization mass cytometry was used to acquire fine lipid fingerprints of BEAS-2B, A549, and H1299 cell lines. After OzID reaction cleaved the C=C double bonds in unsaturated lipids, abundant diagnostic fragment ions were observed. Figure 6 (B, D, F) Specifically, in m / zCharacteristic fragment ion peaks appeared at 566.3089, 622.4078, 650.4391, and 678.4704. Analysis using OzID coupled with electrospray ionization mass spectrometry (ESI-MS / MS) detected a total of 63 C=C isomers. Figure 7 , Figure 8 , Figure 9 , Figure 10 Therefore, the combination of OzID and electrospray ionization mass cytometry can obtain a fine lipid fingerprint of single cells, detecting approximately three times more lipid C=C isomers. Compared with cells not treated with OzID, the fragment ions derived from these reactions significantly enhance the spectral discrimination ability. It should be noted that some lipid compounds produce OzID fragment ions with overlapping signals, such as PC(16:0 / 18:1). m / z 760.5851) contains Δ8, Δ9, and Δ11 isomers, which respectively produce m / z Fragment ions at 636.4235, 650.4391, and 678.4704; similarly, PC(16:0 / 16:1) (m / z 732.5538) contains Δ7 and Δ9 isomers, producing... m / z Fragment ions at 622.4078 and 650.4391. Despite some spectral overlap, the core innovation of this method lies in the fact that the OzID reaction transforms indistinguishable isomer information into quantitatively distinguishable fragment ions. This transformation effectively amplifies biologically relevant differences in isomer composition into a diagnostic fingerprint, thereby achieving more effective cell type differentiation.

[0050] Statistical analysis was performed using statistical software to analyze the high-throughput single-cell mass cytometry results of lung cells BEAS-2B, lung cancer cells A549, and lung cancer cells H1299 before and after the OzID reaction. The resulting UMAP plots ( Figure 6 The results (G) showed that without OzID reaction, the lipid fingerprint profiles of lung cells BEAS-2B, lung cancer cells A549, and lung cancer cells H1299 were not well distinguishable. However, after OzID reaction, the fine lipid fingerprint profiles of these three cells could be effectively distinguished. Furthermore, Figure 6 The results of H show the ion m / z The content of 566.3089 in lung cancer cells A549 and H1299 was significantly higher than that in lung cells BEAS-2B. p <0.005), while ions m / zThe content of 678.4704 in lung cancer cells A549 and H1299 was significantly lower than that in lung cells BEAS-2B. p The value <0.005 indicates that this method can rapidly and accurately identify different types of cells and provides an important methodological basis for the precise structural analysis of lipid markers in cancer.

[0051] Example 4: Fine lipid fingerprinting and differentiation of HL-60 and Jurkat leukemia cells 4.1 Cell Culture HL-60 and Jurkat leukemia cells were cultured separately in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C in a humidified incubator with 5% CO2, and passaged every 2-3 days. When the cell density reached 80%-90%, the cells were collected by centrifugation (1000 rpm, 3 min) and washed twice with phosphate-buffered saline (PBS). After centrifugation again (1000 rpm, 3 min) to remove the PBS, the cells were resuspended in 140 mM ammonium formate aqueous solution containing 20% ​​methanol (pH=7.3), and the cell density was controlled to be approximately 5 × 10⁶ cells / year by cell counting. 4 Cell suspensions of HL-60 leukemia cells and JURKAT leukemia cells were obtained by dividing the cells / mL.

[0052] 4.2 Obtaining mass spectrometric fingerprints and single-cell pulse chromatograms by high-throughput single-cell mass cytometry analysis. Referring to the method in 3.2 of Example 3, high-throughput single-cell mass cytometry analysis was performed on cell suspensions of HL-60 leukemia cells and Jurkat leukemia cells to obtain their basic mass spectrometry fingerprint and fine lipid fingerprint before and after OzID treatment.

[0053] 4.3 Experimental Results Experimental results are as follows Figure 11 As shown, where Figure 11 Figures A through D show the mass spectrometry fingerprints of HL-60 and Jurkat leukemia cells before and after OzID treatment. Figure 11 E represents different cell types and their differences in intercellular lipid characteristics as shown by UMAP analysis after OzID reaction. Figure 11 As can be seen from the data in the middle section, statistical analysis of the high-throughput single-cell mass cytometry analysis results of HL-60 and Jurkat leukemia cells before and after the OzID reaction showed that the OzID reaction had a more significant distinguishing effect. Figure 11 F represents the HL-60 and Jurkat cell diagnostic ion after the OzID reaction. m / z 636.4235 and m / z A comparison chart of expression levels (690.4704). Figure 11 The results of the F test show characteristic diagnostic ions. m / z 566.3089 and m / z The content of 690.4704 in Jurkat leukemia cells was significantly higher than that in HL-60 leukemia cells. p <0.005). This method can not only be used to rapidly identify different cell types, but also accurately quantify the proportion of different lipid isoforms in cells, and has broad application prospects in related fields.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A method of disease single cell high-throughput fine lipidomics analysis, characterized by It comprises the following steps: (1) The cell sample to be tested is prepared into a cell suspension using a methanol-ammonium formate mixed solution, analyzed using electrospray mass spectrometry flow, the analysis results are recorded, and the basic lipid fingerprint of the cell to be tested is obtained; (2) The OzID reaction is carried out by passing O3 into the suspension prepared in step (1), and then analyzed using electrospray mass spectrometry flow after the reaction is completed, the analysis results are recorded, and the fine lipid fingerprint of the cell to be tested is obtained; (3) The analysis results obtained in steps (1) and (2) are compared, and the disease cell type and subtype are distinguished, the disease lipid metabolism regulation mechanism and the disease target are researched through statistical analysis and identification of the single cell basic lipid fingerprint and the fine lipid fingerprint.

2. The disease single cell high-throughput fine lipidomics analysis method according to claim 1, characterized in that: The sample to be tested in step (1) is a disease cell cultured in vitro and a disease primary cell.

3. The disease single cell high-throughput fine lipidomics analysis method according to claim 1, characterized in that: The methanol-ammonium formate mixed solution in step (1) is prepared by adding ammonium formate aqueous solution into methanol; The concentration of the above-mentioned ammonium formate is 100-200 mM; The volume fraction of methanol in the methanol-ammonium formate mixed solution in step (1) is 0-40%; preferably 20%.

4. The disease single cell high-throughput fine lipidomics analysis method according to claim 1, characterized in that: The concentration of the cell sample, cell suspension in step (1) is 3-8 x 10 4 cells / mL.

5. The disease single cell high-throughput fine lipidomics analysis method according to claim 1, characterized in that: The specific steps of the electrospray mass spectrometry flow in steps (1) and (2) are as follows: Cell suspension is pumped at a rate of 1 μL / min through a 25 μm inner diameter capillary via a precision flow rate or pressure control pump. A 1.5 kV DC voltage is applied at the conductive interface connecting the capillary and the pump to create a stable electrospray ionization (ESI) for mass spectrometry detection. The mass spectrometry is performed in full scan mode, with a scanning mass range of... m / z =100~1000, quality resolution is 70000.

6. The disease single cell high-throughput fine lipidomics analysis method according to claim 1, characterized in that: The reaction conditions in step (2) are as follows: O3 flow rate is 0.1-0.5 L / min, and reaction time is 10-30 seconds.

7. The application of the disease single cell high-throughput fine lipidomics analysis method according to any one of claims 1-6 in distinguishing disease cell types and subtypes.

8. The application of the disease single cell high-throughput fine lipidomics analysis method according to any one of claims 1-6 in detecting the proportion of different isomers of lipids in cells.

9. Use according to claim 7, characterized in that It comprises the following steps: The disease single cells are subjected to high-throughput fine lipidomics analysis, the fine lipid fingerprints of different types of disease cells and subtypes are recorded, the different types of disease cells and subtypes are distinguished through statistical analysis, and the lipid markers of different types of disease cells and subtypes are obtained through mass spectrometry identification.

10. A method of disease single cell high throughput identification, characterized in that It comprises the following steps: The known cell lines are detected by the disease single cell high-throughput fine lipidomics analysis method according to any one of claims 1-6, the characteristic fingerprint is recorded, and then the cell to be tested is detected, compared with the characteristic fingerprint, and the identification result is obtained.

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