A single-cell cycle labeling method applicable to organic mass spectrometry and its application in single-cell cycle identification

Abstract

The present invention discloses a method for cell cycle labeling and identification based on organic mass spectrometry detection technology, belonging to the technical field of cell biology. Two types of small molecule tags, namely DNA-targeting molecules and microtubule-targeting molecules, are used to label cells, and the optimized labeling conditions can label living cells. Such tags have good compatibility with organic mass spectrometry detection technology, are applicable to various ionization modes of organic mass spectrometry analysis, have high dissociation efficiency during the ionization process, and good mass spectrometry response. The tag signals and intracellular endogenous lipid signals can simultaneously serve as the basis for cell cycle differentiation and identification, and have good differentiation and identification effects on the G0 / G1, S, G2, and M cell cycle stages. While identifying the single-cell cycle, this method can collect single-cell metabolite information, and further study cell metabolism-related activities at specific cell cycle stages, and has broad application prospects in the fields of stem cell analysis, tumor diagnosis, and systems biology research.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and particularly relates to a single-cell cycle labeling method applicable to organic mass spectrometry and its application in single-cell cycle identification. Background Art

[0002] The cell cycle is ubiquitous in the cells of higher organisms and can generally be divided into two parts: the mitosis phase (M phase) and the interphase. According to the DNA synthesis situation, the interphase can be further divided into three periods, namely the gap 1 phase (G1 phase), the synthesis phase (S phase), and the gap 2 phase (G2 phase). Cells in different cell cycles have significant differences in physical morphology and internal chemical composition: cells in the M phase have significant morphological changes, such as chromosome condensation, nuclear membrane breakdown, spindle appearance, and cell division into two; during the interphase, the morphological structure of cells changes little, but active life activities are carried out inside, and the contents of related enzymes, proteins, nucleic acids, small molecule metabolites, etc. are constantly changing.

[0003] The cell cycle distribution has always been an important indicator of cell proliferation activities. In recent years, with the continuous in-depth study of single cells, the cell cycle stage is more considered to be one of the main sources of cell heterogeneity, and the differences in cell contents caused by cycle differences cannot be ignored. Data obtained without distinguishing cell cycles usually lead to biased research results due to cycle randomness and the averaging of population data. Therefore, identifying the cycle stage of single cells has become the basis for studying various cell life activities and is of great significance in studying cell aging and lesions, cell-drug action mechanisms, and specific cell metabolic pathways.

[0004] In the prior art, the main methods for cell cycle identification mainly include fluorescence label labeling combined with fluorescence imaging / flow cytometry analysis technology, and antibodies labeled with rare metals combined with inorganic mass cytometry technology. The main problems of the fluorescence label combined with fluorescence imaging / flow cytometry analysis technology are that the commonly used DNA labels have poor ability to distinguish between the G2 phase and the M phase, and the fusion protein-based fluorescence labels require relatively complex cell pretreatment and are difficult to be used for real-time analysis of unknown cell samples. Due to the overlap of fluorescence bandwidths, the number of channels for single measurement of fluorescence-based analysis technology is limited. Therefore, it is difficult to monitor other intracellular targets while identifying the cell cycle by fluorescence-based technology. The antibodies labeled with rare metals combined with inorganic mass cytometry technology utilize the advantages of high mass spectrometry resolution and simultaneous detection of multiple substances, and use DNA tags containing rare metals and cell cycle-related antibodies labeled with rare metals as the basis for cycle identification. It can accurately identify each cycle and can simultaneously monitor the changes of dozens of proteins in cells, providing rich molecular information for the study of cell behaviors and disease mechanisms related to proteins in specific cycles. However, this type of inorganic mass spectrometry-based technology is difficult to be used for the detection of intracellular small molecule metabolites. In the research targeting single-cell metabolites, the existing labeling methods and detection technologies cannot achieve single-cell cycle stage identification while detecting single-cell metabolites, and it is still difficult to accurately locate the metabolic behavior of single cells to a specific cycle. Summary of the Invention

[0005] Organic mass spectrometry is the most important tool for single-cell metabolite research. The applicable cell cycle labeling methods and identification methods need to be compatible with cell metabolism analysis and organic mass spectrometry detection at the same time, including: maintaining cell viability during the identification process, having little interference with cell metabolic activity, dissociating during the organic mass spectrometry ionization process, and having strong organic mass spectrometry signal response. Currently, there is still a lack of single-cell cycle labeling and identification methods applicable to organic mass spectrometry detection.

[0006] To solve the problems in the prior art, the first object of the present invention is to provide a single-cell cycle labeling method applicable to organic mass spectrometry detection, including the following steps:

[0007] Obtain a DNA tag molecule stock solution and a microtubule tag molecule stock solution;

[0008] Obtain a single-cell suspension sample, and the single-cell suspension sample includes: a synchronized cycle single-cell suspension sample and a single-cell suspension sample of unknown cycle to be measured;

[0009] Label the synchronized cycle single-cell suspension sample and the single-cell suspension sample of unknown cycle to be measured, including the following two situations:

[0010] The first situation, when the synchronized cycle single-cell suspension sample is not labeled:

[0011] Add the microtubule tag molecule stock solution and the DNA tag molecule stock solution to the synchronized cycle single-cell suspension sample, mix well, and incubate in the dark on a shaker to obtain the labeled single-cell suspension A1;

[0012] Add the microtubule tag molecule stock solution and the DNA tag molecule stock solution to the single-cell suspension sample of the unknown cycle to be measured, mix well, and incubate in the dark on a shaker to obtain the labeled single-cell suspension B1;

[0013] In the second case, when the synchronized cycle single-cell suspension sample has been labeled, that is, the labeled single-cell suspension A1 has been obtained; only the single-cell suspension sample of the unknown cycle to be measured needs to be labeled:

[0014] Add the microtubule tag molecule stock solution and the DNA tag molecule stock solution to the single-cell suspension sample of the unknown cycle to be measured, mix well, and incubate in the dark on a shaker to obtain the labeled single-cell suspension B1;

[0015] Among them, the DNA tag molecule has cell membrane penetrability, DNA targeting ability, and DNA non-covalent binding ability;

[0016] The microtubule tag molecule has the ability to specifically recognize cell microtubules and the ability of non-covalent binding to microtubules.

[0017] Optionally, the concentration of the single-cell suspension sample is 1 - 5×10 6 cells per milliliter.

[0018] Optionally, after labeling the synchronized cycle single-cell suspension sample and the single-cell suspension sample of the unknown cycle to be measured, it further includes the steps of cell washing and redispersion, specifically:

[0019] Centrifuge the labeled single-cell suspension A1 and the labeled single-cell suspension B1 at room temperature respectively, discard the supernatant, obtain cell precipitates, then wash with phosphate buffer solution by centrifugation 3 - 5 times, and then redisperse with an aqueous solution of a volatile substance isotonic to the cells, with a pH of 7.0 - 7.6, to obtain the labeled single-cell suspension A2 and the labeled single-cell suspension B2 with a concentration of 1 - 5×10 4 cells per milliliter respectively.

[0020] Optionally, the DNA tag molecule includes Hoechst33342, Hoechst33258, and the concentration of the DNA tag molecule stock solution is 0.1 - 10 mg / mL.

[0021] Optionally, the microtubule tag molecule includes paclitaxel, docetaxel, and the concentration of the microtubule tag molecule stock solution is 0.1 - 10 mg / mL.

[0022] Optionally, the mass volume ratio of the DNA label molecule to the single cell suspension sample is 0.5-5:1 (μg / mL), the mass volume ratio of the microtubule label molecule to the single cell suspension sample is 0.5-5:1 (μg / mL), and the incubation time is 5-10 min.

[0023] The second object of the present invention is to provide an application of the above method in single cell cycle identification, comprising the following steps:

[0024] S1: Single cell organic mass spectrometry detection: the labeled single cell A2 suspension and the labeled single cell B2 suspension are introduced into an organic mass spectrometry ion source by spotting or injection, and mass spectrometry detection is performed;

[0025] The volume of the sample is controlled at 1-10 μL, and the volume of the cell sample is adjusted to ensure that at most 1 cell is covered during each sampling process;

[0026] The injection flow rate is controlled at 0.5-5 μL / min, and the cell injection concentration is adjusted to ensure that a pulse signal of single cell dispersion is obtained in the ion flow chromatogram detected by mass spectrometry;

[0027] S2: Single-cell mass spectrometry data acquisition: Select the mass spectrometry acquisition mode, set the mass spectrometry detector parameters, and collect single-cell data;

[0028] The number of sampling points in the spotting mode should be greater than 1000, and the injection mode acquisition time should be greater than 30 minutes; each cell sample should collect more than 1000 single-cell data;

[0029] S3: Establishment of single cell cycle differentiation model: Extract and summarize the single cell signals obtained from the synchronous cycle cell samples, and use the clustering algorithm to divide the synchronous cycle cells into 4 categories using the DNA label signal intensity, microtubule label signal intensity, and cell endogenous lipid signal intensity as variables. The 4 types of cells are classified into G0 / G1, S, G2, and M cycle stages according to the sample source;

[0030] S4: Identification of the cycle of the cell samples to be tested: extract and summarize the single cell signals obtained from the unknown cycle cell samples to be tested, and use the DNA label signal intensity, microtubule label signal intensity, and cell endogenous lipid signal intensity as variables. Use a clustering algorithm to assign the single cells of the unknown cycle cell samples to be tested to the G0 / G1, S, G2, and M cycle stage models established in step S3, and identify the cycle stage of each cell according to the assignment results.

[0031] Optionally, the single cell signals in S3 and S4 are extracted from an ion current chromatogram, and a peak signal with a signal-to-noise ratio greater than 10 in the ion current chromatogram is a single cell signal.

[0032] Optionally, the DNA tag signals described in S3 and S4 are the m / z values of their parent ions and daughter ions. Ion current chromatograms are extracted based on these m / z values, with a tolerance error for m / z within 5 ppm. The signal intensities taken in S3 and S4 should be exactly the same.

[0033] Optionally, the intracellular endogenous lipid signals described in S3 and S4 are the m / z values of the parent ions and daughter ions of single or multiple endogenous cell phospholipids between m / z 700 - 900. Ion current chromatograms are extracted based on these m / z values, with a tolerance error for m / z within 5 ppm. The signal intensities taken in S3 and S4 should be exactly the same.

[0034] Optionally, the microtubule tag signals described in S3 and S4 are the m / z values of their parent ions and daughter ions. Ion current chromatograms are extracted based on these m / z values, with a tolerance error for m / z within 5 ppm. The signal intensities taken in S3 and S4 should be exactly the same.

[0035] Advantages of the present invention:

[0036] The cell cycle labeling reagent adopted by the present invention is a class of simple, efficient, and low-cost small molecule reagents, which are suitable for both live cell labeling and organic mass spectrometry detection. It has the characteristics of live cell membrane penetration and non-covalent binding, and shows efficient dissociation effects and extremely high signal responses in organic mass spectrometry detection. Such labeling has the advantages of low cytotoxicity and low metabolic interference at appropriate incubation concentrations and times.

[0037] The present invention can accurately and efficiently distinguish and identify the cell cycle at the single cell level. The cell cycle identification indicators adopted include DNA marker signals, microtubule marker signals, and intracellular endogenous signals that are significantly related to the cell cycle. These signals are used as the basis for cell cycle differentiation at the same time, which can significantly enhance the accuracy and specificity of cell cycle identification. Moreover, using small molecule markers and intracellular endogenous substances as cycle identification indicators can minimize the interference of the cycle identification process on the cell's own activities as much as possible.

[0038] This cell cycle labeling and identification method combined with organic mass spectrometry detection can obtain single cell metabolome molecular information while achieving cell cycle identification, and thus can study cell metabolism-related activities at specific cell cycle stages, having broad application prospects in fields such as stem cell analysis, tumor diagnosis, and systems biology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification, which are used together with the following specific embodiments to explain the present invention, but do not constitute a limitation to the present invention. In the drawings:

[0040] Figure 1Schematic diagram of the method for cell cycle labeling and identification based on organic mass spectrometry according to an exemplary embodiment of the present invention;

[0041] Figure 2 Schematic diagram of the composition of a single-cell flow ion source mass spectrometry device according to an exemplary embodiment of the present invention;

[0042] Figure 3 Partial total ion current chromatogram of Hela cells and extracted ion current chromatograms of m / z 425.208±0.002, m / z 830.336±0.002, and m / z 782.567±0.002 according to an exemplary embodiment of the present invention;

[0043] Figure 4 Classification and PCA results of Hela cells after G0 / G1, S, G2, and M cycle synchronization according to an exemplary embodiment of the present invention;

[0044] Figure 5 PCA results of the cycle attribution of unsynchronized Hela cells according to an exemplary embodiment of the present invention;

[0045] Figure 6 Heat map of metabolite contents of Hela cells in different cycles according to an exemplary embodiment of the present invention;

[0046] Figure 7 Detection results of the activity of Hela cells during the label labeling process according to an exemplary embodiment of the present invention;

[0047] Figure 8 Results of the interference of the label labeling process on the metabolic activity of Hela cells according to an exemplary embodiment of the present invention. Detailed implementation manners

[0048] The technical solutions in the detailed implementation manners will be clearly and completely described below. Based on the detailed implementation manners of the present invention, all other detailed implementation manners obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0049] The single-cell cycle labeling method applicable to organic mass spectrometry in the detailed implementation manners of the present invention includes the following steps:

[0050] Obtaining a DNA tag molecule stock solution and a microtubule tag molecule stock solution: Dissolve a DNA labeling molecule with cell membrane permeability, DNA targeting ability, and DNA non-covalent binding ability in water to prepare a DNA tag molecule stock solution with a concentration of 0.1-10 mg / mL; In some specific implementation manners, the DNA labeling molecule uses Hoechst33342 and Hoechst33258;

[0051] Microtubule marker molecules with cell microtubule-specific recognition and non-covalent binding capabilities are dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 0.1-10 mg / mL; in some specific embodiments, the microtubule marker molecules include paclitaxel (PTX) and docetaxel (DTX).

[0052] Obtaining a single cell suspension sample: The cell suspension sample of the specific embodiment of the present invention includes: a single cell suspension sample of a synchronous cycle and a single cell suspension sample of an unknown cycle to be tested. The single cell suspension sample of the synchronous cycle and the single cell suspension sample of an unknown cycle to be tested are collected separately, centrifuged at room temperature for 3 minutes at 1000 rpm, the supernatant is discarded, and a cell precipitate is obtained. The cells are fully dispersed using a phosphate buffer solution. In some specific embodiments, the concentration of the single cell suspension is adjusted to 1-5×10 6 cells per ml.

[0053] The synchronized cycle single cell suspension should include at least four types of cell samples: synchronized G0 / G1 cycle stage cells (referred to as sample C1), synchronized S cycle stage cells (C2), synchronized G2 cycle stage cells (C3), synchronized M cycle stage cells (C4). Synchronized cells are obtained by in vitro culture and standard cell synchronization experiments as a benchmark for cell cycle identification; unknown cycle waiting cell samples (C x ) can be obtained from multiple sources such as in vitro culture and tissue extraction. For each cell sample, 3-6 parallel samples should be prepared, i.e. C 1-1 , C 1-2 , ..., C x-1 , C x-2 , C x-3 .

[0054] Incubate cells with DNA and microtubule labeling solution at the same time for cell labeling: add PTX or DTX stock solution to each 1 mL of synchronized cycle single cell suspension and unknown cycle single cell suspension to make the final concentration of PTX or DTX in the single cell suspension 0.5-5 μg / mL, mix thoroughly and place in a 37°C shaker in the dark for 5-10 minutes. At the same time, add Hoechst33342 or Hoechst33258 stock solution to the single cell suspension to make the final concentration of Hoechst33342 or Hoechst33258 in the single cell suspension 0.5-5 μg / mL, mix thoroughly and place in a 37°C shaker in the dark for 5-10 minutes to obtain labeled single cell A1 suspension (including labeled synchronized cycle single cell suspension samples C1, C2, C3, C4) and labeled single cell B1 suspension (including labeled unknown cycle to be tested cell sample Cx).

[0055] Cell washing and redispersion: Centrifuge the labeled single-cell A1 suspension and the labeled single-cell B1 suspension at 1000 rpm for 3 min at room temperature, discard the supernatant, wash the cell pellet with phosphate buffer solution by centrifugation 3 - 5 times, centrifuge again at 1000 rpm for 3 min at room temperature, discard the supernatant, redisperse with an aqueous solution of volatile substances isotonic to the cells (pH 7.0 - 7.6), and adjust the concentration of the single-cell suspension to 1 - 5×10 4 cells per milliliter. The finally obtained redispersed single-cell suspension is called LC 1-1 , LC 1-2 , ……, that is, the labeled single-cell A2 suspension; LC x-1 , LC x-2 , LC x-3 , that is, the labeled single-cell B2 suspension, and mass spectrometry detection needs to be completed within 30 min.

[0056] The viability of the finally labeled and washed cells can be detected by a commercial cell viability detection kit, such as the enhanced CCK-8 kit (Beyotime, Shanghai).

[0057] In some specific embodiments, the aqueous solution of volatile substances isotonic to the cells is a 140 mmol / L ammonium formate solution with a pH of 7.3.

[0058] Application of the single-cell cycle labeling method suitable for organic mass spectrometry detection in the specific embodiments of the present invention in single-cell cycle identification includes the following steps, see Figure 1 :

[0059] S1: Single-cell organic mass spectrometry detection: Spot the finally labeled and washed single-cell suspension on a sample target plate or directly inject it into an organic mass spectrometry ion source, and perform mass spectrometry detection.

[0060] Among them, cell loading includes two modes: spotting and injection. The spotting volume of the single-cell suspension is controlled at 1 - 10 μL, and the cell spotting volume is adjusted to ensure that at most 1 cell is covered in each sampling process. The sampling method can be laser sampling or probe sampling; the injection flow rate is controlled at 0.5 - 5 μL / min, and the cell injection concentration is adjusted to ensure a pulsed signal of single-cell dispersion is obtained in the ion current chromatogram of mass spectrometry detection. Peristaltic pumps or syringe pumps can be used for injection.

[0061] Among them, the organic mass spectrometry ion source described in S1 is applicable to laser desorption ionization source, electrospray ionization source, nanoelectrospray ionization source, probe spray ionization source, and single-cell flow ionization source.

[0062] Preferably, a single-cell flow ion source is adopted, which includes an injection pump 1 for cell injection, an injection pump 2 for solvent injection, a four-way connection for mixing cells and solvents, and a nanoliter electrospray ionization needle with a tip of 10-30 μm. The solvent can be a polar organic solvent compatible with mass spectrometry, including methanol, acetonitrile, isopropanol, and their mixed solvents with water. Formic acid can be added to the solvent to assist ionization, and extracellular small molecules can be used as internal standards for cells.

[0063] S2: Single-cell mass spectrometry data acquisition: Select a mass spectrometry acquisition mode and set the parameters of the mass spectrometry detector, and perform single-cell data acquisition under the spotting or injection conditions in S1.

[0064] Among them, the mass spectrometry acquisition modes described in S2 include positive ion mode, negative ion mode, positive / negative switching mode, full scan mode, selected ion monitoring mode, and full scan of the first-level mass spectrometry plus data-dependent second-level mass spectrometry scanning mode.

[0065] Among them, the mass spectrometry detection described in S2 is applicable to conventional mass spectrometry analyzers such as ion trap mass spectrometry, quadrupole mass spectrometry, triple quadrupole mass spectrometry, time-of-flight mass spectrometry, electrostatic field orbitrap mass spectrometry, or Fourier transform ion cyclotron resonance mass spectrometry.

[0066] In some specific embodiments, an electrostatic field orbitrap mass spectrometry is adopted, with a resolution greater than 30000, an acquisition mode of full scan mode in positive ion mode, and an acquisition range of m / z 80-1200.

[0067] In some specific embodiments, the number of sampling points in the spotting mode in S2 should be greater than 1000, and the injection time in the injection mode should be greater than 30 min. Each cell sample should collect more than 1000 single-cell data.

[0068] S3: Establishment of a single-cell cycle discrimination model: Extract and summarize the DNA tag signal intensity, microtubule tag signal intensity, and intracellular lipid signal intensity in the single-cell signals obtained from samples LC1, LC2, LC3, and LC4. Using the DNA tag signal intensity, microtubule tag signal intensity, and intracellular lipid signal intensity as variables, use a clustering algorithm to divide all single cells obtained from samples LC1, LC2, LC3, and LC4 into 4 categories, and classify the 4 categories of cells into G0 / G1, S, G2, and M cycle stages according to the sample source.

[0069] In some specific implementations, the single-cell signals in S3 are extracted from the ion current chromatogram. The peak signals with a signal-to-noise ratio greater than 10 in the ion current chromatogram are considered single-cell signals. The signal intensity values in S3 can be the single-cell peak area, single-cell peak intensity, normalized peak area, normalized peak intensity, peak area after internal standard calibration, and peak intensity after internal standard calibration of each signal in the extracted ion current chromatogram.

[0070] The single cell cycle labeling method suitable for organic mass spectrometry detection according to the specific embodiment of the present invention is used in the single cell cycle identification. The DNA label signal in S3 can take the parent ion m / z and daughter ion m / z of Hoechst33342 and Hoechst33258, and the ion flow chromatogram is extracted based on this m / z value. The tolerance error of m / z is within 5ppm.

[0071] The single cell cycle labeling method suitable for organic mass spectrometry detection according to the specific embodiment of the present invention is used in the single cell cycle identification. The microtubule labeling signal in S3 can take the parent ion m / z and daughter ion m / z of PTX and DTX, and the ion flow chromatogram is extracted based on this m / z value. The tolerance error of m / z is within 5ppm.

[0072] The single cell cycle labeling method suitable for organic mass spectrometry detection according to the specific embodiment of the present invention is used in the single cell cycle identification. The endogenous lipid signal in S3 can take the parent ion m / z and daughter ion m / z of a single or multiple endogenous cell phospholipids between m / z 700-900, and the ion flow chromatogram is extracted based on this m / z value. The tolerance error of m / z is within 5ppm.

[0073] The application of the single cell cycle labeling method suitable for organic mass spectrometry detection in the single cell cycle identification of the specific embodiment of the present invention, the C1, C2, C3, C4 single cell signals used for cluster analysis in S3 are all single cell signals whose DNA label signal intensity, microtubule label signal intensity, and lipid signal intensity are all within the 85%-95% confidence interval in each sample. The remaining single cell signals do not participate in the cluster analysis to ensure the reliability of the clustering results.

[0074] In the application of the single cell cycle labeling method suitable for organic mass spectrometry detection in the specific embodiment of the present invention in the single cell cycle identification, the clustering algorithm in S3 can adopt the K-means algorithm, KNN algorithm, density clustering, hierarchical clustering algorithm, and LDA algorithm.

[0075] In some specific embodiments, the cell cycle differentiation results in S3 can be reduced in dimension and visualized through PCA, tSNE, and Diffusionmaps.

[0076] S4. Identification of cell cycle of the tested cell samples: Extract and summarize the sample LC x The DNA label signal intensity, microtubule label signal intensity, and lipid signal intensity in the obtained single cell signal were used as variables to calculate the LC x The obtained single cells were assigned to the G0 / G1, S, G2, and M cycle phase models established in S3, and the cycle phase of each cell was identified according to the assignment results.

[0077] Application of the single - cell cycle labeling method applicable to organic mass spectrometry detection in the specific implementation manner of the present invention in single - cell cycle identification. In S4, single - cell signals are extracted from the ion current chromatogram, and peak signals with a signal - to - noise ratio greater than 10 in the ion current chromatogram are considered single - cell signals.

[0078] Application of the single - cell cycle labeling method applicable to organic mass spectrometry detection in the specific implementation manner of the present invention in single - cell cycle identification. In S4, the selection of DNA tags, microtubule tags, endogenous lipid signals and their signal intensities is consistent with that in S3.

[0079] Application of the single - cell cycle labeling method applicable to organic mass spectrometry detection in the specific implementation manner of the present invention in single - cell cycle identification. The algorithm in S4 is consistent with that in S3.

[0080] It can be understood that the LC in S4 x A large number of other intracellular signals obtained in the single - cell can be used for the differential analysis of metabolic activities in specific cell stages.

[0081] In addition, it should be noted that the single - cell suspension C without labeling x The single - cell metabolite signals obtained after the same detection conditions as in S1 and S2 can be compared with the metabolite signals obtained by LC in S4 x for differential analysis to verify the interference of the tags and the labeling process on cell metabolic activities.

[0082] Examples

[0083] The Hela cells synchronized in G0 / G1, S, G2, and M cycles are analyzed by using the cell cycle labeling method and the organic mass spectrometry detection method of the present invention. Three groups of Hela cells cultured normally without cycle synchronization are used. Hoechst33258 and DTX are used as DNA tag molecule and microtubule tag molecule respectively, and the signals of Hoechst33258 at m / z 425.208, DTX at m / z 830.336, and phospholipid PC(34:1) at m / z 782.567 are used as cycle discrimination and identification indexes.

[0084] The detection instrument used is a single - cell flow ion source combined with an electrostatic field orbitrap mass spectrometer. The single - cell flow ion source includes the following parts (such as Figure 2As shown in the figure: 1) The injection pump 1 is used for single-cell suspension sampling; 2) The injection pump 2 is used for solvent sampling; 3) The solvent flow path is divided into two paths through a three-way joint. The two solvent flow paths and the cell flow path converge through a micro four-way joint for single-cell extraction. The flow path pipes are all made of polyether ether ketone pipelines with an inner diameter of 65 μm and an outer diameter of 1 / 32″; 4) The outlet end of the micro four-way joint is connected to a stainless-steel zero-dead-volume two-way joint and a nanoelectrospray needle (the needle is a quartz glass capillary; inner diameter 50 μm × outer diameter 150 μm; tip outer diameter 30 μm). The stainless-steel zero-dead-volume two-way joint is connected to the mass spectrometry electrical contact. This part of the device is installed on the commercial nanoelectrospray ion source bracket of an electrostatic field orbitrap mass spectrometer (Q Exactive plus, Thermo Scientific, USA). The mass spectrometry acquisition mode is the positive ion full scan mode (Positive; Full scan), and its working parameters are set as follows: Scan range: 80 - 1200 m / z; Resolution: 35000; Microscans: 1; AGC target: 1e6; Maximum inject time: 50 ms; Sheath gas flow rate: 0; Aux gas flow rate: 0; Sweep gas flow rate: 0; Spray voltage: 3 kV; Capillary temperature: 320 °C; Tube lens voltage: 60 V.

[0085] The specific steps for Hela cell labeling, mass spectrometry detection, and cycle identification are as follows:

[0086] (1) Dissolve Hoechst33258 in water to prepare a 1 mg / mL stock solution, and dissolve DTX in DMSO to prepare a 1 mg / mL stock solution. The stock solutions are stored at -20 °C.

[0087] (2) Collect Hela cells (C 1-1 ,C 1-2 ,C 1-3 ,C 2-1 ,C 2-2 ,C 2-3 ,C 3-1 ,C 3-2 ,C 3-3 ,C 4-1 ,C 4-2 ,C 4-3 ) that are synchronized in the G0 / G1, S, G2, and M phases and three groups of Hela cells (C x-1 ,C x-2 ,C x-3 ,C y-1 ,Cy-2 , C y-3 , C z-1 , C z-2 , C z-3 ), a total of 7 kinds of samples. For each kind of sample, 3 plates of cells were collected in parallel, resulting in a total of 21 tubes of cell samples. The collected cells were centrifuged at 1000 rpm for 3 min at room temperature, and the supernatant was discarded to obtain cell pellets. The cells were fully dispersed using phosphate buffer solution, and the concentration of the single-cell suspension was adjusted to 10 6 cells per milliliter.

[0088] (3) Add 0.6 μL of DTX stock solution to every 1 mL of the single-cell suspension to make the final concentration of DTX in the cell suspension 0.6 μg / mL. After thorough mixing, it was placed in a shaker at 37 °C and incubated for 5 min. Then add 25 μL of Hoechst33258 stock solution to the cell suspension to make the final concentration of Hoechst33258 in the cell suspension 25 μg / mL. After thorough mixing, it was placed in a shaker at 37 °C and continued to be incubated in the dark for 5 min.

[0089] (4) The labeled Hela cells were centrifuged at 1000 rpm for 3 min at room temperature, and the supernatant was discarded. The cell pellets were washed 3 times by centrifugation with PBS, and then centrifuged again at 1000 rpm for 3 min at room temperature. The supernatant was discarded, and the cells were dispersed with 140 mmol / L ammonium formate solution at pH 7.3, and the concentration of the single-cell suspension was adjusted to 2×10 4 cells per milliliter. The labeled Hela cell samples included Hela cells synchronized in the G0 / G1 phase (LC 1-1 , LC 1-2 , LC 1-3 ), Hela cells synchronized in the S phase (LC 2-1 , LC 2-2 , LC 2-3 ), Hela cells synchronized in the G2 phase (LC 3-1 , LC 3-2 , LC 3-3 ), Hela cells synchronized in the M phase (LC 4-1 , LC 4-2 , LC 4-3 ) and three groups of Hela cells that were normally cultured without cell cycle synchronization (LC x-1 , LC x-2 , LC x-3 , LC y-1 , LC y-2 , LC y-3 , LC z-1 , LC z-2 , LC z-3 ).

[0090] (5) The injection flow rate of the single-cell suspension is 1 μL / min. Methanol is used as the solvent, and 36 nmol / L rhodamine B is added as an internal standard in the solvent. The injection flow rate of the solvent is 4 μL / min. The detection time for each single-cell suspension sample is 40 min, and the full-scan mass spectrometry diagram within 40 min is collected.

[0091] (6) Process and summarize the mass spectrometry data of the LC 1-1 ,LC 1-2 ,LC 1-3 ,LC 2-1 ,LC 2-2 ,LC 2-3 ,LC 3-1 ,LC 3-2 ,LC 3-3 ,LC 4-1 ,LC 4-2 ,LC 4-3 samples. The mass spectrometry ion current diagram is as shown in Figure 3 :

[0092] In the total ion current chromatogram of the mass spectrometry of each sample, select the peak signals with a signal-to-noise ratio of the peak intensity greater than 10 as single-cell signals, and record the corresponding chromatographic peak elution time;

[0093] Extract the ion current chromatograms of 4 m / z values from the total ion current chromatogram of the mass spectrometry: m / z 425.208 ± 0.002, m / z 830.336 ± 0.002, m / z 782.567 ± 0.002, m / z 443.233 ± 0.002. Corresponding to the single-cell chromatographic peaks in the total ion current diagram according to the chromatographic peak elution time, and integrate and record the single-cell chromatographic peak area;

[0094] In the mass spectrometry results of each sample, calibrate the chromatographic peak areas of m / z 425.208 ± 0.002, m / z 830.336 ± 0.002, and m / z 782.567 ± 0.002 with the chromatographic peak area of m / z 443.233 ± 0.002 at the same elution time. Retain the single cells with the calibrated peak areas of all three within the 95% confidence interval;

[0095] Summarize the chromatographic peak areas of m / z 425.208 ± 0.002, m / z 830.336 ± 0.002, and m / z 782.567 ± 0.002 after calibration for all cells in each group of samples. Some of the single-cell data are listed in Table 1;

[0096] Table 1 Single-cell data for four cycles

[0097]

[0098] (7) Establish a cell cycle discrimination model: Using the calibrated peak areas of m / z 425.208±0.002, m / z 830.336±0.002, and m / z 782.567±0.002 as three variables, and applying the hierarchical clustering algorithm with the method of Ward's method, the calculation interval being the squared Euclidean distance, and the clustering member being a single solution = 4, classify the cells of LC1, LC2, LC3, and LC4 into the G0 / G1, S, G2, and M cycle stages to establish a cell cycle discrimination model for subsequent identification of cells with unknown cycles. The classification results are visualized through PCA, as Figure 4 shown.

[0099] (8) Cycle identification of three groups of unsynchronized Hela cells (LC x-1 , LC x-2 , LC x-3 , LC y-1 , LC y-2 , LC y-3 , LC z-1 , LC z-2 , LC z-3 ) cultured under normal conditions:

[0100] Summarize the calibrated peak area values of the three groups of Hela cells cultured under normal conditions according to the same data processing steps in (6);

[0101] Mix the calibrated peak area values of the three groups of Hela cells cultured under normal conditions with the model cells in (6), and refer to the hierarchical clustering algorithm in (7) to classify the three groups of Hela cells cultured under normal conditions into the G0 / G1, S, G2, and M cycle stages to achieve the identification of the cycles of unknown single cells. The identification is visualized through PCA, as Figure 5 shown.

[0102] (9) Compare the metabolite composition and content of Hela cells in the range of m / z 80 - 1200 at known different cell cycle stages, and observe significant heterogeneity in metabolite content between cycles, as Figure 6 shown.

[0103] (9) Supplementary verification of cell viability during the labeling process:

[0104] Wash and disperse the C 1-1 , C 1-2 , C 1-3 collected in (2) with reference to (4);

[0105] Examine the cell viability with reference to the detection process of a commercial enhanced CCK-8 kit (Beyotime, Shanghai), and the test results are as Figure 7As shown, the cell viability of the labeled cells remained good in ammonium formate, and more than 80% of the cell viability was maintained within 40 min of mass spectrometry detection.

[0106] (10) Supplementary verification of the interference of metabolic activity in the labeling process:

[0107] Take the C collected in (2) 1-1 , C 1-2 , C 1-3 Wash and disperse with reference to (4);

[0108] Cell sample C 1-1 , C 1-2 , C 1-3 Perform mass spectrometry detection with reference to (5);

[0109] Process the mass spectrometry data and summarize the calibrated peak areas of single cells with reference to (6);

[0110] Summarize the calibrated peak areas of C 1-1 , C 1-2 , C 1-3 and the calibrated peak areas of LC 1-1 , LC 1-2 , LC 1-3 and perform differential verification in PCA. The results are as Figure 8 shown, and no significant difference was observed between the labeled and unlabeled cells.

Claims

1. A single-cell cycle labeling method applicable to organic mass spectrometry detection, characterized in that, It includes the following steps: Obtain a DNA tag molecule stock solution and a microtubule tag molecule stock solution; Obtain a single-cell suspension sample, and the single-cell suspension sample includes: a synchronized-cycle single-cell suspension sample and an unknown-cycle single-cell suspension sample to be tested; Label the synchronized-cycle single-cell suspension sample and the unknown-cycle single-cell suspension sample to be tested, including the following two situations: The first situation, when the synchronized-cycle single-cell suspension sample is not labeled: Add the microtubule tag molecule stock solution and the DNA tag molecule stock solution to the synchronized-cycle single-cell suspension sample, mix well, and incubate in the dark on a shaker to obtain a labeled single-cell A1 suspension; Add the microtubule tag molecule stock solution and the DNA tag molecule stock solution to the unknown-cycle single-cell suspension sample to be tested, mix well, and incubate in the dark on a shaker to obtain a labeled single-cell B1 suspension; The second situation, when the synchronized-cycle single-cell suspension sample is already labeled, that is, a labeled single-cell A1 suspension has been obtained; only the unknown-cycle single-cell suspension sample to be tested needs to be labeled: Add the microtubule tag molecule stock solution and the DNA tag molecule stock solution to the unknown-cycle single-cell suspension sample to be tested, mix well, and incubate in the dark on a shaker to obtain a labeled single-cell B1 suspension; Among them, the DNA tag molecule is a small molecule with live cell membrane penetrability, DNA targeting ability, and DNA non-covalent binding ability; The microtubule tag molecule is a small molecule with cell microtubule specific recognition ability and microtubule non-covalent binding ability; The DNA tag molecule includes Hoechst33342 and Hoechst33258; The microtubule tag molecule includes paclitaxel and docetaxel.

2. The method according to claim 1, wherein The concentration of the single-cell suspension sample is 1 to 5×10 6 cells per milliliter.

3. The method according to claim 2, wherein After the above-mentioned labeling of the synchronized-cycle single-cell suspension sample and the unknown-cycle single-cell suspension sample to be tested, it also includes the steps of cell washing and redispersion, specifically: Centrifuge the labeled single-cell A1 suspension and the labeled single-cell B1 suspension at room temperature respectively, discard the supernatant to obtain cell pellets, then centrifuge and wash with phosphate buffer solution 3 to 5 times, and then redisperse with an aqueous solution of a volatile substance isotonic to the cells, with a pH of 7.0 to 7.6, to respectively obtain a labeled single-cell A2 suspension and a labeled single-cell B2 suspension with a concentration of 1 to 5×10 4 cells per milliliter.

4. The method according to claim 1, wherein The concentration of the DNA tag molecule stock solution is 0.1 - 10 mg / mL.

5. The method according to claim 1, characterized in that The concentration of the microtubule tag molecule stock solution is 0.1 - 10 mg / mL.

6. The method according to claim 1, wherein The mass-volume ratio of the DNA tag molecule to the single-cell suspension sample is: 0.5 - 5:1 (μg / mL), the mass-volume ratio of the microtubule tag molecule to the single-cell suspension sample is: 0.5 - 5:1 (μg / mL), and the incubation time is 5 - 10 min.

7. Use of the method according to claim 1 in single cell cycle identification, characterized in that, It includes the following steps: S1: Single-cell organic mass spectrometry detection: Respectively introduce the labeled single-cell A2 suspension and the labeled single-cell B2 suspension into the organic mass spectrometry ion source by spotting or injection, and perform mass spectrometry detection; Among them, the volume of the spotting is controlled at 1 - 10 μL, and the cell spotting volume is adjusted to ensure that each sampling process covers at most 1 cell; The flow rate of the injection is controlled at 0.5 - 5 μL / min, and the cell injection concentration is adjusted to ensure a pulsed signal of single-cell dispersion in the ion flow chromatogram of the mass spectrometry detection; S2: Single-cell mass spectrometry data acquisition: Select the mass spectrometry acquisition mode, and set the mass spectrometry detector parameters to acquire single-cell data; Among them, the number of sampling points in the spotting mode should be greater than 1000, and the sampling time in the injection mode should be greater than 30 min; each cell sample should collect more than 1000 single-cell data; S3: Establishment of single-cell cycle discrimination model: Extract and summarize the single-cell signals obtained from the synchronized cycle cell samples, and use the DNA tag signal intensity, microtubule tag signal intensity, and intracellular endogenous lipid signal intensity as variables to classify the synchronized cycle cells into 4 categories using a clustering algorithm, and classify the 4 categories of cells into the G0 / G1, S, G2, and M cycle stages according to the sample source; S4: Identification of the cycle of the cell sample to be tested: Extract and summarize the single-cell signals obtained from the cell sample to be tested with an unknown cycle, and use the DNA tag signal intensity, microtubule tag signal intensity, and intracellular endogenous lipid signal intensity as variables. Use a clustering algorithm to assign the single cells of the cell sample to be tested with an unknown cycle to the G0 / G1, S, G2, and M cycle stage models established in step S3, and identify the cycle stage of each cell according to the attribution result.

8. The application according to claim 7, wherein The single-cell signals described in S3 and S4 are extracted from the ion current chromatogram, and the peak signal with a signal-to-noise ratio greater than 10 in the ion current chromatogram is the single-cell signal.

9. The application according to claim 7, wherein The DNA tag signal described in S3 and S4 is its parent ion m / z and daughter ion m / z. The ion current chromatogram is extracted with this m / z value, and the tolerance error of m / z is within 5 ppm. The signal intensities taken in S3 and S4 should be exactly the same.

10. The application according to claim 7, characterized in that, The intracellular endogenous lipid signal described in S3 and S4 is the parent ion m / z and daughter ion m / z of one or more intracellular endogenous cell phospholipids between m / z 700 - 900. The ion current chromatogram is extracted with this m / z value, and the tolerance error of m / z is within 5 ppm. The signal intensities taken in S3 and S4 should be exactly the same.

11. The application according to claim 7, wherein The microtubule tag signal described in S3 and S4 is its parent ion m / z and daughter ion m / z. The ion current chromatogram is extracted with this m / z value, and the tolerance error of m / z is within 5 ppm. The signal intensities taken in S3 and S4 should be exactly the same.

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