A single-cell online extension lysis mass spectrometry flow analysis method

Abstract

The application discloses a single-cell online prolonging lysis mass spectrometry flow analysis method. A cell suspension sample is injected into a separation capillary, a front section of the capillary is spirally wound to realize single dispersion of the cells, and a part of a rear section of the capillary is located in an ultrasonic device to realize online ultrasonic lysis, localized single-cell lysis contents are obtained, the content solution gradually enters a non-contact electrospray needle to realize ionization, and finally enters a mass spectrometry detector for detection. Meanwhile, the analysis coverage of weakly polar compounds can be improved by combining a plasma generation device. The method can realize single-cell analysis with high throughput and high metabolite coverage, and by means of a prolonged single-cell detection window, the collection of metabolite primary mass-to-charge ratio information and the identification of secondary structures under high resolution can be realized, including qualitative and quantitative analysis of positional isomers, so that the method has wide application prospects in the fields of cell identification, cell typing, differential substance research, tumor diagnosis and biomarker mining.

Description

Technical Field

[0001] This invention relates to an extended lysis mass cytometry technique for analyzing single cells, including a cell monodisperse method, a universal single-cell ultrasonic lysis method, a non-contact electrospray ionization method, and a single-cell level metabolite and isomer detection and quantification method based on mass spectrometry detection intensity, and their applications in single-cell identification and typing. Background Technology

[0002] Cells are the smallest functional units of living organisms. Traditional biological techniques typically study large populations of cells. While this approach has laid the foundation for existing biological theories, averaged experimental results overlook the crucial cellular heterogeneity, posing a significant obstacle to accurately understanding the physiological processes of each individual cell and obscuring information about rare cell populations. Therefore, independent analysis and measurement of each cell can not only help reveal the true physiological processes of cells under specific environments, such as aging, differentiation, and signal transduction, but also allow for non-discriminatory analysis of rare cell populations. This is crucial for a comprehensive understanding of cell population classification, the discovery of relevant cell subtypes under specific disease contexts, and the identification of disease targets. However, due to the small size of individual cells, their extremely limited contents (only fL-pL in normal somatic cells), low but diverse metabolites, and varied isoforms with a wide dynamic range, existing life science analytical techniques face significant challenges in terms of sensitivity, analytical throughput, dynamic range, and structural identification capabilities.

[0003] Mass spectrometry, as an analytical tool with high sensitivity, high throughput, wide detection range and good qualitative capabilities, plays a very important role in the field of single-cell metabolite analysis. Based on this, existing single-cell metabolite analysis methods are mainly divided into two categories according to analytical throughput and metabolite coverage. One category mainly uses micromanipulation to aspirate or extract contents, followed by mass spectrometry analysis. Although this type of method has low cell analysis throughput and is difficult to operate, it has high metabolite coverage and strong qualitative and absolute quantitative capabilities. Representative methods can be found in the literature (B. Shrestha, A. Vertes. Anal. Chem., 2009, 81, 8265-8271 and N. Pan, W. Rao, N. R. Kothapalli, R. Liu, A. W. G. Burgertt and Z. Yang. Anal. Chem., 2014, 86, 9376-9380). Combined with the ionization mode of induced spraying, the spraying time can be further extended. Representative methods can be found in the literature (H. Zhu, G. Zou, N. Wang, M. Zhuang, W. Xiong and G. Huang. Proc. Natl. Acad. Sci. USA, 2017, 114, 2586-2591; another type of method involves monodispersing cells and then sequentially detecting them by mass spectrometry. Cell integrity is maintained during the separation process. Although this method has high analytical throughput and is relatively simpler to operate, the rupture process only occurs instantaneously during the electrospray process, resulting in a very narrow single-cell detection window. Metabolite coverage, including structural identification and quantification capabilities, is lacking. Representative methods can be found in the literature (Yao, H., H. Zhao, X. Zhao, X. Pan, J. Feng, F. Xu, S. Zhang and X. Zhang. Anal. Chem., 2019, 91, 9777-9783. and Xu, S., M. Liu, Y. Bai and H. Liu. Angew. Chem. Int. Ed. 2021, 60, 1806-1812.). Therefore, while ensuring high cell analysis throughput, how to extend the single-cell detection time as much as possible to detect more intracellular endogenous metabolites and thus improve structural identification capabilities is an urgent problem to be solved in the future of single-cell metabolomics analysis methods. Summary of the Invention

[0004] To achieve high-throughput cell analysis while extending the single-cell detection window, thereby improving the acquisition resolution of primary mass-to-charge ratio data and the qualitative ability of secondary metabolite structures, including the differentiation of structural isoforms, and to overcome the problems of short single-cell detection time, low cell analysis throughput, insufficient metabolite coverage, and lack of secondary structure identification capability in existing methods, this invention aims to provide a universal, ultrasound-assisted online single-cell extended lysis mass spectrometry analysis method. This method extends the single-cell detection window, improves metabolite coverage, enhances the accuracy of primary mass-to-charge ratio data, obtains secondary structure information at the single-cell level, and combines characteristic fragments to perform qualitative and quantitative analysis of the single-cell expression of isoforms.

[0005] The present invention first provides an online extended lysis mass cytometry analysis device for single-cell analysis, comprising a cell monodispersing device, an ultrasonic device, a non-contact electrospray needle, and a mass spectrometer detector. The cell monodispersing device is a separating capillary, the front section of which is spirally wound, a portion of which is located in the ultrasonic device, and the tail section is connected to the non-contact electrospray needle, which is aimed at the inlet of the mass spectrometer detector.

[0006] Cell suspension is injected into one end of the separation capillary, and single cells are dispersed through the separation capillary. Then, ultrasonic cavitation causes the flowing cells to undergo an online lysis process. The other end of the capillary is connected to a non-contact electrospray needle. Finally, the cell contents are ionized in the needle section by non-contact electrospray, and then entered into mass spectrometry for detection.

[0007] In the aforementioned online extended lysis mass cytometry analyzer for single-cell analysis, the cell monodisperse device can be a quartz capillary with an inner diameter of 150-250 μm, an outer coating of polyimide, and a spiral winding section with 5-8 turns and a spiral diameter of 6-10 cm. After the cell suspension is injected into the cell monodisperse device, the shear stress gradient generated under the action of Dean's flow microfluidic flow in the spiral winding section will sort and separate the cells in the cell suspension, thereby obtaining a monodisperse cell arrangement. The number of spiral turns and the diameter directly affect the magnitude of the shear force of Dean's flow, thus affecting the cell dispersion effect.

[0008] The ultrasonic device can be an ultrasonic cleaner with a power of 50-200W and an ultrasonic frequency of 40KHz. After passing through a capillary immersed in an ultrasonic ice bath, monodisperse cells undergo accelerated cell membrane rupture and content release due to ultrasonic cavitation, ultimately forming a localized content solution. The length of the capillary subjected to ultrasonic treatment is generally 10-15cm.

[0009] The non-contact electrospray nozzle is generally made of a capillary tube with the coating removed, connected to a separating capillary tube via a constant-diameter two-way valve. The inner diameter of the nozzle tip is 30-60 μm, and a DC induced electric field is applied to the outer wall of the nozzle by winding copper wire. Under the action of the induced electric field, the electrospray process is formed, and the analyte enters the mass spectrometer detector in ion form.

[0010] The mass spectrometer detector is suitable for determinations using ion trap mass spectrometry, quadrupole mass spectrometry, triple tandem quadrupole mass spectrometry, time-of-flight mass spectrometry, electrostatic orbital trap mass spectrometry, and / or Fourier transform ion cyclotron resonance mass spectrometry.

[0011] Based on the above-mentioned device, this invention provides an ultrasound-assisted online extended lysis mass spectrometry flow cytometry analysis method for single cells, which includes steps such as preparation of cell suspension, cell sample introduction and monodispersion, ultrasonic treatment, non-contact electrospray ionization and mass spectrometry detection, and data processing. Specifically, as follows:

[0012] 1) Prepare cell suspension samples C1, C2, ..., C at specific concentrations. x , where x is a natural number representing the cell sample number;

[0013] 2) The cell suspension sample was injected into the cell monodisperse device in the above-mentioned online extended lysis mass spectrometry flow cytometry analysis device through a syringe, and the cells were subjected to online ultrasonic lysis and mass spectrometry detection;

[0014] 3) Data analysis, including: extracting multiple step-like total ion current maps of single cells with a continuous analysis time greater than 10 s from the mass spectrometry detection results obtained in step 2); selecting the mass spectrum with the highest total ion current intensity as the analytical mass spectrum for that cell; normalizing based on the total ion current intensity to obtain the relative expression intensity of metabolites in each cell; and obtaining the expression distribution of different isoforms within a single cell by comparing the intensity of characteristic secondary mass spectrometry peaks of isoforms based on the single-cell secondary mass spectrometry data. This data can serve as the basis for subsequent cell typing and metabolite differential analysis.

[0015] Step 1) above prepares the cell suspension. The cells in the logarithmic growth phase collected by centrifugation are washed to remove salts and then resuspended in a dispersion to obtain the cell suspension sample to be tested.

[0016] Preferably, the concentration of the cell suspension sample to be tested is 1000-10000 cells / mL, and the cell sample to be tested can be cells from a single cell line, cells from a mixture of multiple cell lines, or an unknown cell suspension sample, etc.

[0017] In step 1), the cells can be washed with an 80% methanol aqueous solution. The final dispersion used to disperse the cells is an organic solution with cell fixation properties (such as an aqueous methanol solution), preferably an ice-cold 80% methanol aqueous solution.

[0018] Step 2) is used for online ultrasonic lysis and mass spectrometry detection of cells. The specific analysis process of cell samples using the above-mentioned online extended lysis mass spectrometry flow cytometry analysis device is as follows: The prepared cell suspension is delivered to the spirally wound capillary section by the action of a syringe pump. After the cells are monodispersed, they enter the ultrasonic device. Ultrasonic cavitation in an ice-water bath promotes cell lysis, thereby obtaining localized single-cell lysate contents. The contents solution gradually enters the non-contact electrospray nozzle, where ionization is achieved under the action of an induced electric field. Finally, the mass spectrometry detector is used for detection, recording the mass ion flow map changing over time and the mass spectrum acquired at each moment. The induced electric field can be achieved by applying a DC high voltage to the copper wire wound around the nozzle using a commercially available nanoliter electrospray ionization source.

[0019] Preferably, the injection volume in step 2) is 0.2-0.5 mL each time, and the injection flow rate is set to 0.5-3 μL / min.

[0020] The preferred ultrasonic conditions in step 2) are an ice-water bath, with the capillary immersed in the ice-water bath for a length of 10-15 cm.

[0021] The induced voltage applied to the non-contact electrospray nozzle is generally a DC high voltage, which can be in positive or negative mode, and the voltage value can be set to 2-4kV. The applied voltage can also be a pulsed high voltage, which can be in positive or negative mode, with a peak value of 2-4kV and a frequency of 10-10000Hz.

[0022] The preferred mass spectrometer for acquisition is a high-resolution mass spectrometer with a full scan resolution greater than 30,000. The acquisition mode is either a full scan or a full scan / secondary alternating scan mode. The secondary scan targets the top seven strongest ions in the full scan. The acquisition range is 100-1500 m / z. Each data acquisition session lasts for more than 60 minutes, and the mass spectrometry information of 2-3 cells can be acquired per minute.

[0023] Because endogenous metabolites are diverse and vary widely in volatility and polarity, combining two or more ionization methods can broaden the scope of metabolite analysis and further improve the coverage of single-cell metabolite identification. Electrospray ionization (ESI) is primarily advantageous for detecting small molecules with high polarity and volatility, but it remains limited for low-polarity small molecule metabolites and lipids, and suffers from severe ion inhibition. Penning ionization based on plasma can analyze compounds with moderate or weak polarity, complementing the analytes applicable to ESI ionization. Plasma is generated by corona discharge of a working gas (such as helium, nitrogen, or argon) in a re-discharge chamber. The excited-state atoms in the plasma instantaneously desorb and ionize compounds in the sample, enabling mass spectrometry detection. This technique requires no solvent, has no inhibition effect, exhibits excellent detection capability for non-polar substances, and has a simple apparatus. Combining two ionization methods can broaden the scope of single-cell metabolite identification and, as a post-ionization method, further improve ionization efficiency, thereby enhancing method sensitivity. The specific procedure involves setting up a plasma generator to post-ionize the electrospray ionized sample, followed by mass spectrometry detection (see [link to documentation]). Figure 7 The plasma emission direction generated by the plasma generator forms a certain angle (15-90 degrees) with the line connecting the tip of the non-contact electrospray nozzle and the inlet of the mass spectrometer detector. Simultaneously, a mechanical vacuum pump is installed at the inlet of the mass spectrometer detector to ensure the vacuum level at the inlet. The plasma generator can employ plasma sources including, but not limited to, Direct Real-Time Analysis (DART), Low-Temperature Plasma (LTP), Dielectric Barrier Discharge (DBDI), and Flowing Atmospheric Pressure Afterglow (FAPA).

[0024] In one embodiment of the present invention, an MCF-7 cell suspension sample with a concentration of 10,000 cells / mL was analyzed. The injection flow rate was 1 μL / min. The cell monodispersing device was a quartz capillary with an outer diameter of 360 μm and an inner diameter of 150 μm, coated with polyimide, and the spiral section had 6 turns with a diameter of 8 cm. The length of the separation capillary immersed in the ultrasonic ice bath was 15 cm. The outlet was connected to a capillary electrospray needle with the coating removed via a constant diameter tee. The needle tip had an inner diameter of 50 μm and an outer diameter of 150 μm. A -3 kV DC induced electric field was applied through copper wire wound around the tube wall. An Orbitrap mass spectrometer was used for detection, with a full scan resolution of 35,000 and a secondary scan resolution of 17,500. The acquisition range was 100-1500 m / z, and the acquisition time was 60 min. The obtained mass spectra showed an average pulse duration greater than 20 s, as well as a large number of intracellular metabolites with a mass-to-nucleus ratio (m / z) in the range of 100-1500. The structure and intensity information of multiple pairs of isomers were also obtained through secondary spectral acquisition.

[0025] Step 3) above involves data analysis. In the obtained total ion chromatogram, the mass spectrum of the single cell is obtained at the point of highest total ion chromatogram signal intensity in each single-cell pulse. The single-cell mass spectra are then processed as follows:

[0026] 3-1) Extract the signal intensities M1, M2, ..., M of the corresponding metabolites from the spectrum according to the theoretical mass-to-nucleus ratio of the metabolites. b b represents the number of metabolites identified, and the tolerance error for the cytoplasm-to-nucleus ratio is within 5 ppm.

[0027] 3-2) The metabolite signal intensities were normalized using the total ion current intensity S as a reference. The normalized intensities were M1 / S, M2 / S, ..., M b / S, therefore, the mass spectrometry detection results for each cell can be obtained: C x ((M1 / S) x (M2 / S) x , ..., (M b / S) x ), where x represents the cell sample number.

[0028] 3-3) In the single-cell secondary mass spectrum, the characteristic peak intensities of a pair of isomers A are I and I, respectively. a1 ,I a2 Therefore, mass spectrometry detection results of different isoforms in each cell can be obtained: C x ((I a1 / I a2 )x,(I b1 / I b2 )x…(I n1 / I n2) x ), where x represents the cell sample number and n is the number of isoform analyses.

[0029] The mass spectrometry results of multiple cells obtained in steps 3-2) and 3-3) can be summarized into a data matrix:

[0030]

[0031] By utilizing the normalized intensity values ​​of variables detected in single cells (i.e., each variable in the aforementioned data matrix, including metabolites and isomers), data analysis algorithms (such as principal component analysis, visual clustering, volcano plots, heatmaps, etc.) are used for cell typing, identification, differential analysis, and metabolic pathway research.

[0032] The ultrasound-assisted online extended lysis technique provided by this invention is a highly versatile, high-throughput single-cell analysis technique with strong structural identification and differentiation capabilities. The extended single-cell analysis time not only provides sufficient time for acquiring higher-resolution primary mass-to-charge ratio information but also makes secondary structure identification at the single-cell level possible, thus ensuring the application of this invention's extended lysis method in isoform differentiation and qualitative and quantitative analysis. This ultrasound-assisted online extended lysis technique can be used for cell identification, cell typing, differential metabolite and isoform research, and has broad application prospects in fields such as biomarker mining and tumor diagnosis.

[0033] The device for cell dispersion in this invention is a simple and low-cost flow cytometry device with functions of cell sample introduction, cell dispersion and arrangement. It can be manufactured with capillaries of different inner and outer diameters and has good compatibility with ultrasound devices. It can achieve online lysis and analysis of 2-3 cells per minute. Moreover, this device is versatile, not limited to cell types, and can be adapted to various types of mass spectrometry analyzers.

[0034] The ultrasonic device used for cell lysis in this invention can be a commonly used ultrasonic cleaner in the laboratory. The device is simple and inexpensive, and the ultrasonic process can be controlled by adjusting the length of the capillary immersed in the liquid.

[0035] The ionization method used in this invention is non-contact electrospray, which applies a DC-induced electric field by wrapping copper wire around the tube wall. This method is simpler and easier to operate than conventional AC pulse generation equipment. Furthermore, this ionization method eliminates the need for a liquid circuit interface connected to a power tee, thus significantly reducing the likelihood of cell debris clogging the tubing. This is crucial for the parallelism of experiments involving long-term, large-volume cell sample analysis. Adding plasma ionization further expands the detection range of weakly polar substances in single cells, increasing the quantity and variety of metabolites that can be identified in single cells. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the ultrasonic-assisted online extended lysis mass cytometry analysis device used in an embodiment of the present invention, wherein: 1 is the cell suspension inlet; 2 is the single-cell dispersion device, i.e., the separation capillary; 3 is the ultrasonic cleaner; 4 is the non-contact electrospray needle; and 5 is the mass spectrometer detector.

[0037] Figure 2 These are the total ion chromatograms of single cells and the ion chromatograms of related metabolite extraction obtained by extended lysis mass cytometry in this embodiment of the invention, with and without the addition of cells. (a) is the detection result of the cell suspension with cells, and (b) is the detection result of the blank solvent without cells.

[0038] Figure 3 The images show single-cell pulses and background mass spectra obtained from prolonged lysis mass cytometry analysis of MCF-7 cells in this embodiment of the invention.

[0039] Figure 4 The three pairs of isomers identified by secondary spectral comparison in MCF-7 cells in this embodiment of the invention and their content distribution in single cells are as follows: a) the differential characteristic fragment ions of 2-isopropylmalic acid and 3-isopropylmalic acid and their relative content distribution in 12 single cells; b) the differential characteristic fragment ions of 4-phenylbutyric acid and eugenol and their relative content distribution in 12 single cells; c) the differential characteristic fragment ions of 4-hydroxybutyric acid and 3-hydroxybutyric acid and their relative content distribution in 12 single cells.

[0040] Figure 5 The above is a visualization clustering result of five types of tumor cells based on the signal intensity after metabolite normalization in this embodiment of the invention.

[0041] Figure 6 This is a visualized clustering result of MCF-7 cells further refined by combining isoform information in an embodiment of the present invention.

[0042] Figure 7This is a schematic diagram of an ultrasound-assisted online extended lysis mass cytometry analyzer for single cells, combining ESI and plasma post-ionization. The components are: 1, cell suspension inlet; 2, single-cell dispersion device (separation capillary); 3, ultrasonic cleaner; 4, non-contact electrospray needle; 5, mass spectrometer detector; 6, plasma generator; and 7, mechanical vacuum pump. Detailed Implementation

[0043] The technical solution of the present invention will be further illustrated below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited by the specific conditions of these embodiments.

[0044] Example: The ultrasound-assisted single-cell online extended lysis technology of the present invention was used to analyze the metabolic activities of five types of tumor cells, and the intracellular isoform of MCF-7 cells was structurally identified and quantitatively analyzed.

[0045] The online extended pyrolysis mass cytometry analysis device used is as follows: Figure 1 As shown, the cell suspension enters the single-cell dispersion device 2 under the action of the injection pump. It first enters the spirally wound separation capillary section, and the single cells are gradually dispersed under the action of Dean flow microfluidics. The monodispersed cells then enter the capillary section immersed in the ice-water bath of the ultrasonic cleaner 3, and the single cells are lysed online by ultrasound. The cell contents obtained by online lysis finally flow to the non-contact electrospray needle 4, where they are ionized under the action of the induced electric field and enter the mass spectrometer detector 5 to complete the subsequent mass spectrometry detection.

[0046] The specific steps are as follows:

[0047] (1) A single-cell dispersion device 2 was fabricated using a quartz capillary with an outer diameter of 360 μm and an inner diameter of 150 μm. The spiral winding portion had 6 turns and a spiral diameter of 8 cm. The length of the capillary immersed in the ultrasonic chamber of the ultrasonic cleaner 3 was 15 cm, and ice water was placed in the ultrasonic chamber. One end of the capillary was connected to a syringe as the cell suspension inlet 1, and the other end was connected to a non-contact electrospray needle 4 with its coating removed. The outer wall of the needle was wrapped with copper wire, and the inner diameter of the needle tip was 50 μm and the outer diameter was 150 μm. The connected device was installed in front of the inlet of the Orbitrap mass spectrometer detector 5, with the needle 4 mm away from the inlet of the mass spectrometer detector 5.

[0048] (2) Wild-type MCF-7 cells cultured adherently in 3.5 cm cell culture dishes were used as the analysis subject. When cells were in good growth condition, they were washed once with PBS, then digested with trypsin, and the suspended cells were collected and counted using a cell counter. The cells were washed twice with 80% cold methanol solution, and finally diluted to 10⁻⁶ with ice-cold 80% methanol solution. 3 / mL, to obtain cell suspension.

[0049] (3) Take 1 mL of cell suspension and immediately perform online single-cell lysis analysis. That is, the single cells are first dispersed through the separation capillary, and the dispersed single cells are sonicated in an ice bath to achieve online release of cell contents. Then, they flow through a non-contact electrospray needle to achieve electrospray ionization under the action of an induced electric field, and finally achieve mass spectrometry detection. The parameters of mass spectrometry flow cytometry analysis are set as follows: injection flow rate 1 μL / min, voltage value -4 kV, acquisition mode full scan and full scan / secondary alternating scan mode, full scan mass spectrometry resolution set to 35000, secondary scan resolution set to 17500, acquisition range m / z 100-1500, and acquisition time 60 min.

[0050] (4) Pulse mass spectra of single cells were extracted from the collected ion chromatograms. Figure 2 The figures show the total ion chromatograms corresponding to the three cell pulses and the extracted ion chromatograms of two endogenous metabolites (pyruvate and deoxyribose). Compared to the ultrasound results showing no obvious total ion pulse peaks and no ion pulse peaks for endogenous metabolites in the blank solvent, the ultrasound results of the cell suspension showed obvious pulse peaks with a pulse duration of approximately 20 seconds. This indicates that the pulse peaks were not introduced by the ultrasound process but were related to cell sample introduction. Therefore, this invention significantly extends the time for online single-cell detection, while maintaining a cell analysis throughput of 2-3 cells / min.

[0051] Similarly, using this method, a total of 141 MCF-7 single-cell pulse peaks were acquired, including those from single-cell pulses and background mass spectra ( Figure 3 Compared to the background spectrum, the cell pulses showed a significant increase in characteristic mass spectrometry peaks of endogenous metabolites (mainly short-chain and long-chain fatty acids). Furthermore, the extended analysis time facilitated high-resolution mass spectrometry acquisition. A richer array of peaks was observed in single-cell pulses, containing a large number of short-chain and long-chain fatty acids. Figure 3 The magnified images show the peak shapes of leucine and dimethylsphingosine at acquisition resolutions of 35,000 and 280,000. Using a higher resolution acquisition mode yields narrower peak shapes, thus improving the confidence level of primary information identification of metabolites in single cells. A total of 213 metabolites were identified in negative mode, with a cytoplasm-to-nucleus ratio error tolerance set to within 5 ppm. These 213 metabolites are designated as M1, M2, ..., M... 213 Three pairs of isomers, 2-isopropylmalic acid and 3-isopropylmalic acid, 4-phenylbutyric acid and eugenol, and 4-hydroxybutyric acid and 3-hydroxybutyric acid, were identified using secondary information. The secondary characteristic fragment ion mass spectrometry responses are represented as I... a1 / I a2 Ib1 / I b2 I c1 / I c2 ( Figure 4 Compared to the changes in the content of the first two pairs of isomers (within two-fold), the relative changes in the content of 4-hydroxybutyric acid (4-HYBA) and 3-hydroxybutyric acid (3-HYBA) spanned a 100-fold, thus potentially indicating further potential for cell subtype differentiation. The intensities of metabolites extracted from each mass spectrum were normalized to the total ion current intensity S, yielding the following data matrix (other tumor cell types were treated similarly):

[0052]

[0053] Based on the normalized signal intensity of each cell metabolite as a variable, a visual clustering method was used to successfully distinguish two types of breast cancer cells (MDA-MB-231, MCF-7), one type of cervical cancer cell (HeLa), one type of rectal cancer cell (HCT116), and one type of osteosarcoma cell (U2OS). Figure 5 The single-cell prolonged lysis mass cytometry method of this invention can effectively distinguish five types of tumor cells based on the obtained metabolite information, demonstrating the method's good typing ability. Regarding isomers, [the method is described in the original text]. Figure 4 It was observed that the content changes of the first two pairs of isomers were within 2-fold, while the content changes of the last pair of isomers spanned several hundred-fold. This may suggest differential expression of 4-hydroxybutyric acid (4-Hydroxybutyric acid) and 3-hydroxybutyric acid (3-HHB) in MCF-7 cells. Qualitative and quantitative isomer analysis is a capability currently lacking in other online single-cell metabolite analysis technologies. Without isomer dimension information, only one cell cluster was observed in the MCF-7 cell clustering results. After incorporating the isomer dimension into cell typing, further fine-tuning of the cells was performed through visualization of the clustering results. MCF-7 cells were divided into three cell clusters, as shown below. Figure 6 As shown, this further illustrates the significant differences in the expression of this pair of isoforms among different MCF-7 cells. Figure 6 ).

Claims

1. A single-cell online extended lysis mass cytometry analysis method, comprising the following steps: 1) Prepare cell suspension samples C1, C2, ..., C with a concentration of 1000-10000 cells / mL. x , where x is a natural number representing the sample number; 2) The cell suspension sample is injected into a cell monodisperse device using a syringe. The cell monodisperse device is a separating capillary with a helical winding at the front and a portion of the rear section located within an ultrasonic device. The tail end is connected to a non-contact electrospray nozzle, which is aligned with the inlet of the mass spectrometer detector. After entering the cell monodisperse device, the cells are monodispersed in the helical winding section, and then subjected to online ultrasonic lysis via the ultrasonic device to obtain localized single-cell lysate. The lysate solution gradually enters the non-contact electrospray nozzle, where it is ionized under the influence of an induced electric field. Finally, mass spectrometry is performed using a full scan / alternating two-stage scanning mode. The cell monodisperse device uses a quartz capillary with an inner diameter of 150-250 μm, and the helical winding section has 5-8 turns with a diameter of 6-10 cm. The ultrasonic device is an ultrasonic cleaner with a power of 50-200 W and an ultrasonic frequency of 40 kHz. The length of the capillary subjected to ultrasonic treatment is 10-15 cm. cm, immersed in an ice water bath; the inner diameter of the tip of the non-contact electro-spray nozzle is 30-60 μm, and the outer wall of the nozzle is wrapped with copper wire for applying direct current; 3) Perform data analysis on the mass spectrometry detection results obtained in step 2), including: extracting multiple step-shaped total ion current maps of single cells with a continuous analysis time of more than 10 s, taking the mass spectrum with the highest total ion current intensity as the analysis mass spectrum of the cell, normalizing according to the total ion current intensity to obtain the relative expression intensity of metabolites in each cell; and obtaining the expression distribution of different isoforms in a single cell by comparing the intensity of characteristic secondary mass spectrometry peaks of isoforms based on the single-cell secondary mass spectrometry data.

2. The single-cell online extended lysis mass cytometry analysis method as described in claim 1, characterized in that, In step 1), the logarithmic growth phase cells collected by centrifugation are washed and desalted, then resuspended in a dispersion to obtain a cell suspension sample with a concentration of 1000-10000 cells / mL.

3. The single-cell online extended lysis mass cytometry analysis method as described in claim 1, characterized in that, Step 2) Each injection volume is 0.2-0.5 mL, and the injection flow rate is 0.5-3 μL / min; when the mass spectrometer detector performs detection, it records the mass ion flow chromatogram that changes over time and the mass spectrum acquired at each time step.

4. The single-cell online extended lysis mass cytometry analysis method as described in claim 1, characterized in that, In step 2), a plasma generator is added to post-ionize the electrospray ionized sample before mass spectrometry detection. A mechanical pump is installed at the inlet of the mass spectrometer detector to ensure the vacuum level at the inlet.

5. The single-cell online extended lysis mass cytometry analysis method as described in claim 1, characterized in that, Step 3) In the obtained total ion chromatogram, obtain the mass spectrum of the single cell at the point of highest total ion chromatogram signal intensity in each single-cell pulse, and perform the following data processing on the single-cell mass spectrum: 3-1) Extract the signal intensities M1, M2, ..., M of the corresponding metabolites from the spectrum according to the theoretical mass-to-nucleus ratio of the metabolites. b b represents the number of metabolites identified, and the tolerance error for the cytoplasm-to-nucleus ratio is within 5 ppm. 3-2) The metabolite signal intensities were normalized using the total ion current intensity S as a reference. The normalized intensities were M1 / S, M2 / S, ..., M b / S, thus obtaining the mass spectrometry detection results for each cell: C x (M1 / S) x (M2 / S) x , ..., (M b / S) x ), x represents the sample number; 3-3) In the single-cell secondary mass spectrum, the characteristic peak intensities of a pair of isomers A are I and I, respectively. a1 , I a2 Therefore, mass spectrometry detection results of different isoforms in each cell were obtained: C x (I) a1 / I a2 ) x , (I b1 / I b2 ) x …(I) n1 / I n2 ) x x represents the sample number, and n is the number of isomers analyzed.

6. The single-cell online extended lysis mass cytometry analysis method as described in claim 5, characterized in that, The mass spectrometry results of multiple cells obtained in steps 3-2) and 3-3) are summarized into a data matrix: Different cell types are identified, classified, or differentially analyzed based on the normalized intensity of metabolites and isomers in the data matrix.

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