CN113281441A - Single cell organelle mass spectrum detection method and detection device - Google Patents

Abstract

The invention discloses a single organelle mass spectrometry detection method, which comprises a method for obtaining single organelle information by combining single organelle patch clamp technology and mass spectrometry technology. The invention also discloses a single organelle mass spectrometry detection device. The invention combines single lysosome patch clamp technology and electrospray ion source mass spectrometry technology to establish single lysosome mass spectrometry technology. Patch-clamp techniques can detect the activity of ion channels or transporters on lysosomal membranes, while electrospray ionization mass spectrometry can perform timely analysis of metabolic samples. After recording the electrical signal of the lysosome with the lysosomal patch clamp technique, the lysosomal content is sampled for mass spectrometry detection, so as to quantitatively analyze the composition of the lysosomal content, and the sample does not require pretreatment. This method enables simultaneous detection of lysosomal function and metabolic state. Compared with the traditional analysis method of lysosome homogenate, single lysosome mass spectrometry technology can more faithfully reflect the metabolic state of lysosome.

Description

Single cell organelle mass spectrum detection method and detection device

Technical Field

The invention relates to the technical field of organelle analysis, in particular to a mass spectrum detection method and a mass spectrum detection device for a single cell device.

Background

Lysosomes are vesicular organelles coated with a single-layer membrane in eukaryotic cells and can degrade biological macromolecules in the cells. Lysosomes are heterogeneous, so it is essential to develop studies of individual lysosomal metabolism to further reveal the functional regulation of lysosomes. The method for researching the metabolism of the organelles in the prior art is mainly adopted and comprises the steps of separating and purifying the organelles through differential centrifugation or density gradient centrifugation, homogenizing the organelles and then identifying components. The method exposes lysosomes to a non-cellular environment for a long time, which is prone to sample loss and the production of metabolic byproducts. Kelly, b.m. et al found that lysosomes are heterogeneous organelles that vary in morphology, size, activity and function, and this analysis also masked metabolic differences between lysosomal individuals.

Liu, b. et al found that the activity of ion channels or transporters on lysosomal membranes could be detected by simultaneously recording electrical signals on individual lysosomes using the patch clamp technique, but that qualitative and quantitative analysis of individual lysosomal contents was not possible.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a mass spectrum detection method of a single cell device, which solves the technical problem that the prior art cannot effectively detect and analyze the content of an organelle.

In order to solve the technical problems, the invention provides the following technical scheme: a single-cell instrument mass spectrum detection method comprises a method for acquiring single-cell instrument information by combining a single-cell instrument patch clamp technology with a mass spectrum technology.

Further, the unicell device information includes composition information of the content of the single cell.

Further, the single-cell instrument mass spectrum detection method comprises the following steps:

step one, adopting a single-cell device patch clamp technology to obtain the content in a single-cell device;

and step two, performing mass spectrometry detection on the content in the single organelle obtained in the step one.

Further, the unicellular device patch clamp technology of the first step comprises the following steps: sucking the contents in the organelles into the electrode by applying negative pressure to obtain the contents in the organelles; wherein the length of the electrode tip is 5-10mm, and the diameter of the tip opening is 0.1-2 μm.

The length of the tip of the glass microelectrode electrode used in the mass spectrum sampling experiment is 5-10mm, and the diameter of the opening of the tip is 0.1-2 mu m, so as to prevent the sample from being diluted by the external liquid of the electrode; the extra-electrode fluid from mass spectrometry sampling is a cytosolic ion environment that mimics the lysosome. Thus, a spray rate of pL/min can be obtained with high reproducibility and sensitivity for detection of substances, and the detection limits for Lysine (LYS), Histidine (HIS) and Arginine (ARG) are 0.045. mu.M, 0.042. mu.M and 0.038. mu.M, respectively.

Further, the present invention employs NH₄Compared with the traditional electrode solution, the Cl electrode solution improves the signal-to-noise ratio of mass spectrometry detection, and can obtain electrophysiological and mass spectrometry signals simultaneously.

Further, the electrode tip length was 8mm and the tip opening diameter was 0.5 μm.

Further, the unicellular device patch clamp technology of the first step comprises the following steps:

step 1) culturing cells;

step 2) acquiring electrophysiological information of the unicell device of the cells cultured in the step 1);

step 3) extracting the content of the single organelle.

Further, step 2) is to perform organelle enlargement treatment before treating the organelles by using the electrodes.

Further, the cells were treated with vacuolin-1 to effect the enlargement of the lysosome.

Because organelles (e.g., lysosomes, endosomes) are too small to be directly sampled using a glass electrode, the increased handling of organelles facilitates single-organelle mass spectrometry sampling.

Further, the organelles are derived from the following cells: comprises at least one of HEK-293T cells, BxPC-3 cells, mouse cardiac muscle cells, heart fibroblasts, mouse cerebral cortical neurons, glial cells, mouse peritoneal macrophages, mouse embryonic fibroblasts and mouse lymph fibroblasts.

Further, the unicellular device comprises at least one of a monosome, an endosome.

The invention also discloses a single cell mass spectrometer detection device, which comprises a patch clamp and a mass spectrometer, wherein the patch clamp is used for extracting the content in the single cell organelle, and the mass spectrometer is used for carrying out mass spectrometry on the obtained content in the single cell organelle.

The invention has the advantages that: the invention combines the lysosome patch clamp technology and the electrospray ion source mass spectrum technology, and establishes the monosysosome mass spectrum technology. The patch clamp technique can detect the activity of an ion channel or a transporter on a lysosome membrane, and the electrospray ion source mass spectrometry technique can analyze metabolic samples in time. After the electric signal of the lysosome is recorded by the lysosome patch clamp technology, the lysosome content is sampled for mass spectrum detection, so that the content components of the lysosome are quantitatively analyzed, and the sample does not need to be pretreated. The method realizes simultaneous detection of lysosome function and metabolic state. Compared with the traditional analysis method of lysosome homogenate, the monosomic mass spectrometry technology can reflect the metabolic state of lysosome more faithfully.

Drawings

FIG. 1 is a graph of P-values versus relative intensities for lysosomal and cytoplasmic metabolites of the invention.

FIG. 2 is a graph showing the relative intensity distribution of lysosomal metabolites and cytoplasmic metabolites in the present invention.

FIG. 3 is a record diagram of typical current of lysosomes of HEK-293T cells with overexpression EGFP-N1, rPQLC2 and PQLC2 knockout containing 50mM lysine in electrode internal solution respectively.

FIG. 4 is a statistical plot of current density of-100 mV for lysosomes of HEK-293T cells overexpressing EGFP-N1, rPQLC2 and PQLC2, which contain 50mM lysine in the electrode solution, respectively, according to the present invention.

FIG. 5 is a record of typical current of lysosomes of HEK-293T cells with overexpression EGFP-N1, rPQLC2 and PQLC2 knockouts, wherein the electrode solutions of the invention respectively contain 50mM histidine.

FIG. 6 is a statistical plot of current density of-100 mV for lysosomes of HEK-293T cells overexpressing EGFP-N1, rPQLC2 and PQLC2, which contain 50mM histidine in the electrode solution, respectively, in accordance with the present invention.

FIG. 7 is a record of typical current of lysosomes of HEK-293T cells with overexpression EGFP-N1, rPQLC2 and PQLC2 knockouts, wherein the electrode solutions of the invention respectively contain 50mM arginine.

FIG. 8 is a statistical plot of current density of-100 mV for lysosomes of HEK-293T cells overexpressing EGFP-N1, rPQLC2 and PQLC2 containing 50mM arginine in the electrode solutions, respectively, according to the present invention.

FIG. 9 is a mass spectrum of lysine in a single lysosome of the HEK-293T cells of the present invention.

FIG. 10 is a statistical graph of the relative lysine content in individual lysosomes of HEK-293T cells of the invention.

FIG. 11 is a mass spectrum of histidine in a single lysosome of the HEK-293T cells of the present invention.

FIG. 12 is a statistical plot of the relative content of histidine in individual lysosomes of HEK-293T cells of the invention.

FIG. 13 is a mass peak of arginine within a single lysosome of the HEK-293T cells of the invention.

FIG. 14 is a statistical plot of the relative arginine content within individual lysosomes of HEK-293T cells of the invention.

FIG. 15 is a schematic view of the shape of each glass electrode and tip in the present invention.

FIG. 16 is a bar graph of the relative content of lysine detected in the case of glass electrodes of different shapes according to the present invention.

FIG. 17 is a bar graph of the relative content of histidine detected with glass electrodes of different shapes according to the present invention.

FIG. 18 is a bar graph of the relative content of arginine detected with glass electrodes of different shapes according to the present invention.

FIG. 19 is a plot of mass spectra peaks of TIC and SIC of ALF extracted in the present invention. Wherein the spray flow rate is 3.2 pL/min. SIC denotes the selected ion flow; TIC denotes total ion current.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

In this example, ex vivo HEK-293T cells were used as an example, but the present invention can also be applied to cells such as Mouse Embryonic Fibroblasts (MEF), Mouse Lung Fibroblasts (MLF), bladder cancer cells (T24), human bladder epithelial immortalized cells (SV-HUC-1), BxPC-3 cells, mouse cardiac myocytes and cardiac fibroblasts, mouse cerebral cortical neurons and glial cells, mouse peritoneal macrophages, and mouse skin fibroblasts (MEF).

The embodiment discloses a single cell instrument mass spectrum detection method, which comprises the following steps:

step one, culturing cells

Culture of HEK-293T cells

HEK-293T cells for passaging were cultured in T25 flasks. Removing the culture medium in the culture bottle by suction, adding 1mL of preheated trypsin for rinsing once, adding 1mL of trypsin, and placing in a carbon dioxide incubator at 37 ℃ for digestion for 2 min; adding 4mL of preheated culture medium into a culture bottle, stopping digestion, blowing 10 times by using a 1mL pipette gun, and blowing away cells to form cell suspension; a new T25 flask was added with 5mL of pre-heated medium, 250. mu.L of cell suspension was added to the flask, and the flask was shaken obliquely to mix the cells. Cells with a cell density of 5% can grow full, generally for three days, and then passage is carried out.

Among them, the cell culture medium for culturing HEK-293T cells is preferably DMEM (Gibco-derived medium containing various amino acids and glucose) medium containing 10% fetal bovine serum (i.e., fetal bovine serum at a volume ratio of 1:10 to the cell culture medium, derived from Biological Industries) and 1% penicillin-streptomycin (i.e., penicillin-streptomycin diabody at a volume ratio of 1:100 to the cell culture medium, derived from Biosharp). The cells are cultured at 37 deg.C with 5% carbon dioxide (i.e. CO)₂ Body ofVolume 5%) in the incubator.

Further, when the cell density is as high as 90% -98%, passaging can be performed in a biosafety cabinet.

Step two, transfection of plasmids

For transient transfection of HEK-293T cells, Lipofectamine2000 was used for transfection. Plasmids used in mass spectrum sampling experiments, electrophysiological experiments and imaging experiments comprise PQLC2-EGFP, LAMP1-mCherry, LAMP2-EGFP, mChery 2-LC3, EGFP-Rab5A and the like. When co-transfection is involved, the plasmid mass ratio is 1: 1.

The cells of this example were cultured in 35mm dishes, and when HEK-293T cells were grown to a density of 70%, the plasmid of interest was transfected using Lipofectamine 2000. Mu.g of plasmid (1. mu.g of each of the two plasmids, if co-transfected) and 7. mu.L of Lipofectamine2000 were diluted separately in 150. mu.L of Opti-MEM Medium.

The diluted plasmid and Lipofectamine2000 were mixed into the same EP tube and blown up and mixed well. Standing at room temperature for 5 min. Dropwise adding the mixture into a cell culture medium for culturing HEK-293T cells, and incubating for 12h to obtain cells containing lysosome markers.

Then digested with trypsin and plated in imaged chamber at 50% -70% cell density for confocal microscopy imaging experiments. Or spread on a polylysine treated circular cover slip of 12mm diameter. The method is used for electrophysiological experiments and mass spectrometry sampling experiments.

Further, in this example, HEK-293T cells were transiently transfected with mCherry-tagged Lamp1 (i.e., LAMP1-mCherry) or Rab5A (i.e., EGFP-Rab5A) plasmids, labeling lysosomes. Only fluorescently labeled lysosomes were selected for recording and sampling. Primary cells were incubated by lysotracker (invitrogen) to label lysosomes.

Step three, the monosomic patch clamp technology comprises the following steps:

1. cover glass treatment

Treatment of a 12mm diameter round cover glass (ISO 9001):

1) 150mL of tap water and two drops of liquid detergent are added into a 200mL beaker, 500 slides are pulled open and put into water, and the glass slides are shaken for 5-10 min.

2) And (4) washing the glass slide for 5-10min by running water and then washing the glass slide for 30 times by using ultrapure water.

3) The glass slide is soaked in 100mL of hydrochloric acid (5.3mL of concentrated hydrochloric acid with the volume percentage of 36-38% is constant volume to 100mL) for 2 days, and the mouth of the beaker is sealed by a preservative film and tin foil paper.

4) The slides were rinsed 30 times with ultrapure water. The slides were washed 3 times with analytically pure absolute ethanol. Then 100mL of absolute ethyl alcohol is used for immersing the glass slide, the mouth of the beaker is sealed by a preservative film and tin foil paper, and the beaker is placed at room temperature overnight.

5) Pouring off the absolute ethyl alcohol soaked in the glass slide in the beaker, covering the opening of the beaker with tinfoil paper, leaving a small hole for volatilizing the absolute ethyl alcohol, and placing the beaker in a 60 ℃ oven to dry the glass slide.

6) And (5) checking whether the slide is completely processed under a microscope, and then sterilizing the slide by using an autoclave.

7) The sterilized slides were poured into 10cm glass dishes in an intercellular biosafety cabinet and incubated with 0.1mg/mL Polylysine (PLL) for 12h at room temperature.

8) The PLL was recovered, the slide was washed 4 times with sterilized ultrapure water, and then the slide was immersed in ultrapure water and placed in a biosafety cabinet for use.

Preparation of 2.0.1% polylysine solution

Preparation of borate buffer (100 mL): 0.48g of boric acid and 0.25g of sodium borate were dissolved in sterilized ultrapure water, and the volume was adjusted to 100 mL. 100mg of polylysine powder was dissolved in 100mL of borate buffer to prepare a 0.1% polylysine solution. After filtration through a 0.22 μm filter, the resulting mixture was stored in a refrigerator at 4 ℃.

Further, before use, a 0.1% polylysine solution was diluted 10-fold with a borate buffer solution and used.

3. Lysosome enlargement treatment

Cells containing the lysosome-labeled plasmid after transfection of the plasmid of interest in step two were plated on a polylysine-treated slide glass of 12mm diameter and cultured for 12 hours.

After the cells were fully adherent, lysosomes were enlarged by treatment with 1 μ M vacuolin-1 overnight.

vacuolin-1 is a lipid soluble polycyclic triazine that selectively increases endosome and lysosomal size through homotypic membrane fusion.

4. Acquiring electrophysiological data

The contents of the monosome were sampled using borosilicate electrodes. The drawing of the glass electrode for mass spectrum sampling is strict, the drawn electrode tip is 5-10mm long, and the diameter of the tip opening is 0.1-2 μm, and the electrode tip 8mm long and the diameter of the tip opening is 0.5 μm are preferably adopted in the embodiment. The elongated electrode tip may reduce the potential for dilution of the sample by the extra-electrode fluid. The glass electrode is obtained by means of a two-step drawing. The parameters involved in the drawing process are shown in table 1.

Table 1 mass spectrometry sampling glass electrode draw down parameters (Ramp 532)

The acquisition of electrophysiological data specifically comprises the steps of:

4.1) transfer of Vacuolin-1 treated cells to a cell containing 140mM K-gluconate, 4mM NaCl, 2mM MgCl₂、0.39mM CaCl₂And 10mM HEPES (pH 7.2 adjusted with KOH) in a bath of an external electrode solution.

4.2) use the glass electrode tip to cut open the cell membrane and manually separate the enlarged organelles (i.e., lysosomes) from the cells.

The electrode internal liquid comprises the following components: 185mM NH₄HCO₃And 80mM NH₄And (4) Cl. The electrode resistance of the liquid in the filling electrode is 5-16M omega.

4.3) after giga-seals were formed, the film was broken by ZAP pulses (1V, 0.5-2 ms).

4.4) monosomal voltage clamp recording. The voltage stimulus was-70 mV, 2 s; +70mV, 2 s. The voltage holding is 0mV for each sweep 10 s.

Further, the present invention collects only lysosomes with tight seals (>1G Ω) for analysis to ensure that luminal samples are not contaminated by electrode effluent.

5. Obtaining the content of monosomes

After the electrophysiological data were collected, the content composition of the monosome was obtained by applying negative pressure. Whether sampling was successful was confirmed by observing changes in the volume of monosomes. Once intraluminal content is imbibed into the electrode, the volume of the monosome is significantly reduced.

Step four, mass spectrometry

The glass electrode containing the contents of the monosome was quickly removed from the bath and then subjected to qualitative and quantitative analysis of the contents of the monosome by mass spectrometry (MS mass spectrometry).

Further, the invention also discloses the following configurations of the electrode internal liquid and the electrode external liquid:

preparing electrode internal liquid: 0.5218g NH were weighed₄HCO₃The powder was added to 25mL of ultrapure water. An additional 2mL of HCl (1mol/L) was added, and a large amount of bubbles were generated during this process. And removing bubbles generated by the reaction by using an ultrasonic instrument or a vortex instrument for reuse so as to avoid influencing sealing of records and mass spectrum detection. After the preparation, the mixture was filtered through a 0.22 μm filter, and the filtrate was dispensed into an EP tube and stored in a refrigerator at-20 ℃.

Preparation of external electrode solution (500 mL): ultrapure water was added to a beaker, 16.3975g of Kgluconate, 400. mu.L of NaCl (5mol/L mother liquor), 1mL of MgCl were added₂(1mol/L mother liquor), 195. mu.L CaCl₂(1mol/L mother liquor) and 1.1915g HEPES. The pH was adjusted to 7.2 with KOH. Finally, the volume is increased to 500mL by using ultrapure water. The osmolality of the solution was measured using an osmometer. The solution was poured into a 500mL blue-capped bottle and the information on the solution was marked clearly. Storing in a refrigerator at 4 deg.C, filtering and packaging before use.

The invention adopts a mass spectrometry method for the monosome established by combining a monosome spectrum technology and a monosome mass spectrometry technology to detect the metabolite composition of the cytoplasm and the lysosome of HEK-293T cells. As shown in fig. 1 and 2, lysosomes are metabolic organelles, and the content of most metabolites in lysosomes is significantly higher than that in cytoplasm. The lysosome was found to be rich in cystine, valine, choline and cysteine, while the cytoplasm was rich in carnitine, creatine, phosphocholine, aspartic acid, glutamic acid, taurine, glycine and proline, consistent with what the investigators detected with the lysosome homogenate. Also, many other different metabolites were found in the cytosol and lysosome. In which the dots above the horizontal dashed line in figure 1 represent significantly different metabolites. The black areas in fig. 2 indicate the corresponding metabolites are relatively high in intensity.

The invention combines the lysosome patch clamp technology and the electrospray ion source mass spectrum technology, and establishes the monosysosome mass spectrum technology. The patch clamp technique can detect the activity of an ion channel or a transporter on a lysosome membrane, and the electrospray ion source mass spectrometry technique can analyze metabolic samples in time. After the electric signal of the lysosome is recorded by the lysosome patch clamp technology, the lysosome content is sampled for mass spectrum detection, so that the content components of the lysosome are quantitatively analyzed, and the sample does not need to be pretreated. The method realizes simultaneous detection of lysosome function and metabolic state. Compared with the traditional analysis method of lysosome homogenate, the monosomic mass spectrometry technology can reflect the metabolic state of lysosome more faithfully. By establishing a monosomic mass spectrometry method, the metabolic research is expanded from the single cell level to the single organelle level, and a powerful technical means is provided for basic biological research.

Example 2

The invention also discloses a freezing and recovering mode of the cells.

HEK-293T cells can be subcultured cell lines which are generally frozen in liquid nitrogen and then recovered when used. In addition, cells knocked out by the specific gene CRISPR/Cas9 are also stored in liquid nitrogen in a freezing mode. Cultured primary cells are generally not cryopreserved.

Furthermore, the invention also discloses a freezing and recovering mode of the cells.

HEK-293T cells can be subcultured cell lines which are generally frozen in liquid nitrogen and then recovered when used. In addition, cells knocked out by the specific gene CRISPR/Cas9 are also stored in liquid nitrogen in a freezing mode. Cultured primary cells are generally not cryopreserved.

Cell cryopreservation and recovery

The method for freezing and storing the HEK-293T cells comprises the following steps:

1) HEK-293T cells cultured in T75 flasks can be frozen when the density reaches 80% -90%.

2) A50 mL centrifuge tube was prepared, 900. mu.L DMSO (Sigma) was added, and 11.1mL of medium (volume fraction 10% FBS, volume fraction 1% penicillin-streptomycin, and volume fraction 89% DMEM) was added and mixed until needed.

3) HEK-293T cells to be cryopreserved were trypsinized. The medium in the T75 flask was first aspirated and rinsed once with 3mL of pre-warmed trypsin. Then, 3mL of trypsin was added and digested in a 37 ℃ carbon dioxide incubator for 2 min. Finally, 3mL of medium was added to stop digestion and 10 strokes were performed to blow the cells apart to form a cell suspension.

4) 6mL of the cell suspension was pipetted using an electric pipette and added to the 50mL centrifuge tube prepared above. And (4) reversing and mixing.

5) Subpackaging into freezing tubes, and dividing the cell suspension into 36 tubes with 500 mu L/tube. Then the freezing tube is put into a programmed cooling box and stored in a refrigerator at the temperature of minus 80 ℃. The next day, the cells were transferred to liquid nitrogen for storage.

The method for recovering the HEK-293T cells comprises the following steps:

1) a T25 flask was prepared, 5mL of medium (10% FBS by volume, 1% penicillin-streptomycin by volume and 89% DMEM by volume) was added, and the mixture was preheated in a 37 ℃ carbon dioxide incubator.

2) Taking out 1 tube of HEK-293T cells from the liquid nitrogen tank, and quickly thawing in a water bath at 37 ℃.

3) The cell suspension was aspirated, and the cell suspension was added to the previously prepared T25 flask, and the flask was tilted to mix the cells. Culturing in a carbon dioxide incubator at 37 deg.C.

4) After 12h, replacing a new culture medium and continuing culturing.

Example 3

In order to examine the stability and reliability of the method of the present invention, the cationic amino acid transporter PQLC2 on the lysosome membrane was used to transport amino acids.

PQLC2 can transport cationic amino acids in the lysosome into the cytoplasm, and the current generated by PQLC2 mediated cationic amino acid transport was recorded using hololysosome patch-clamp.

As shown in fig. 3-8, when the HEK-293T cells overexpress rplcc 2, compared to the overexpression of EGFP-N1, rplcc 2 was recorded to mediate cationic amino acids as: significant inward currents are produced by lysine, histidine and arginine transport. Since the basal current generated by the background level of PQLC2 mediated cationic amino acid transport was relatively small, there was no significant difference in the current recorded on the lysosomes of EGFP-N1 and PQLC2 knockout HEK-293T cells.

In the figure, 1 represents EGFP-N1 (i.e., lysosome overexpressing EGFP-N1), 2 represents rPQLC2 (i.e., lysosome overexpressing rPQLC 2), and 3 represents PQLC 2-knocked-out HEK-293T (i.e., lysosome of PQLC2-KO or lysosome of PQLC 2-knocked-out HEK-293T). Data in the graph are expressed as mean ± sem, where P <0.001, and numbers within the histogram indicate the number of lysosomes detected.

Meanwhile, HEK-293T cells are subjected to PQLC2 knockout, EGFP-N1 overexpression or rPQLC2 overexpression respectively, then lysosome contents in the cells are detected by a mass spectrometer, and lysine, histidine and arginine in lysosomes are relatively quantitatively analyzed.

As shown in fig. 9-14, the content of lysine, histidine and arginine in the lysosomes was significantly reduced for the over-expressed rpqc 2 relative to the EGFP-N1. The content of lysine, histidine and arginine in the PQLC2-KO is remarkably increased relative to the lysosome with EGFP-N1 over-expression, and here it can also be seen that the mass spectrometric detection is more sensitive than the electrophysiological detection. This result is consistent with the expected result, verifying the reliability established by the analysis method of the present invention.

Data are expressed as mean ± sem, where P <0.01, P <0.001, P < 0.0001. The numbers in the histogram indicate the number of lysosomes detected.

The present embodiment preferably uses CRISPR/Cas9 technology to knock out PQLC2, although other prior art PQLC2 knock-out methods also use the present invention.

The specific experimental steps of the HEK-293T cell PQLC2 knockout scheme are as follows:

1. designing gRNA

The CRISPRgRNA was designed using genome editing tools.

SEQ ID No.1PQLC2-gRNA-T1-fwd:5’-CACCGCTCCTACTCTCTGTTCGCGC-3’

SEQ ID No.2PQLC2-gRNA-T1-rev:5’-AACGCGCGAACAGAGAGTAGGAGC-3’

SEQ ID No.3GCD-Set1-PQLC2-gRNA-T1-fwd:5’-AAGGTGAGGGGCTGAGCTTG-3’

SEQ ID No.4GCD-Set1-PQLC2-gRNA-T1-rev:5’-ACGCTGTGTGCAGGCAGAAG-3’

2. Construction of gRNA into vectors

And (3) mixing gRNA: the SEQ ID No. 55 '-CTCCTACTCTCTGTTCGCGC-3' sequence was constructed into the lentiviral vector lentiCRISPRV 2.

TABLE 2 digestion of the plenti-CRISPR System (60. mu.L)

Digestion was carried out at 37 ℃ for 30 min. The gel recovered a 14kb band.

100mM DTT formulation: 20mL (0.01M sodium acetate pH 5.2) was added 0.309g of DTT powder. 0.016404g of sodium acetate were dissolved in 20mL of water and the pH was adjusted to 5.2 with glacial acetic acid. Then 0.309g DTT powder was added to 0.01M sodium acetate solution, mixed well, protected from light and stored at-20 ℃.

TABLE 3 sgRNA annealing System (10. mu.L)

The reaction was carried out at room temperature for 10 min. Aspirate 4. mu.L of ligation into 50. mu.L of competent cells for transformation, plating (Amp +). After 12h, picking monoclonal shake bacteria at 200rpm for 10 h. And extracting the plasmid by using the rapid miniextraction kit. Sequencing, and the primer is U6-renyuan.

3. Producing virus

1) On the first day, 6cm dishes of HEK-293T cells were plated to achieve a cell density of 50% -80% on the second day. The medium used comprised 10% volume fraction FBS + 90% volume fraction DMEM, cultured overnight.

2) The next day, transfection was performed. Two clean EP tubes were taken, one of which was added with 250. mu.L of Opti-MEM (i.e., Opti-MEM Medium) + 1. mu.g of KO plasmid (plasmid for knocking out PQLC 2) + 1. mu.g of PAX2+ 0.5. mu.g of pMD2.G, and the other was added with 250. mu.L of Opti-MEM + 17. mu.L of lipo2000 (i.e., Lipofectamine2000), and left to stand for 5 min. Then the solutions of the two EP tubes were mixed, blown and mixed well, and then left to stand for 20 min. Then slowly and uniformly dripping into a culture dish of HEK-293T cells. The virus packaging plasmid is PAX₂And pmd2. g.

3) On the third day, after 15 hours, the culture medium volume fraction was changed to 10% FBS + 89% DMEM + 1% double antibody (double antibody, i.e., penicillin-streptomycin), and the culture was continued for 24 hours.

4) On the fourth day, the supernatant was collected, filtered through a 0.45 μm filter into a 15mL centrifuge tube, and dispensed into 0.5mL tubes and stored at-80 ℃.

4. Infected cell

1) On the first day, HEK-293T cells were cultured in 35mm dishes using 10% FBS + 90% DMEM by volume fraction overnight to a cell density of 50% -60% on the next day.

2) The next day, cells were changed. The medium was replaced with DMEM medium containing 10% FBS, 8. mu.g/mL polybrene, 25% virus by volume and incubated overnight for at least 24 h.

3) On the third day, the cells were changed. The medium was changed to DMEM medium containing 10% FBS by volume and puromycin at 3. mu.g/mL and incubated overnight for 24 h. Subculturing and continuing with DMEM medium containing 10% FBS by volume and puromycin at 3. mu.g/mL. Single cell clones were established using limiting dilution until identified.

5. Identification

1) Solution preparation:

1mL Boiling buffer(25mM NaOH，2mM EDTA)＝2.5μL NaOH(10N)+4μL EDTA(0.5M)+993.5μL H₂O。

1mL Neutralizing buffer(40mM Tris，pH 7.4)＝40μL Tris(pH 7.4，1M)+960μL H₂O。

2) the method for extracting the genome from the cell comprises the following steps: cells from a 35mm dish were digested into a suspension and collected in a 1.5mL EP tube. 5000rpm 3min, discard the supernatant. 1mL of 1 XPBS was added to resuspend the cells, and the cells were centrifuged at 5000rpm for 3min, and the supernatant was discarded. Add 100. mu.L of a mounting buffer, metal bath 100 ℃ for 20 min. Vortex for 10s, centrifuge. Adding 100 mu L of neutrallizing buffer, blowing and mixing evenly, and centrifuging at 12000rpm for 2min, wherein the supernatant is the cell genome template.

3) PCR amplification of the fragment of interest:

TABLE 4 PCR reaction System (50. mu.L)

And (3) PCR reaction conditions: at 95 ℃ for 3 min; 95 ℃, 15s, 67 ℃, 15s (set touch down-0.7 ℃/cycle), 72 ℃, 30s, 30 cycles; 72 ℃ for 10 min; at 8 ℃ and infinity.

4) And (4) PCR reaction products are sequenced, and CRISPR knock kout results are identified.

Example 4

The embodiment discloses a single cell mass spectrometer detection device, which comprises a patch clamp and a mass spectrometer, and is characterized in that the patch clamp is used for extracting contents in a single cell organelle, and the mass spectrometer is used for performing mass spectrometry on the acquired contents in the single cell organelle.

Example 5

This example is an example of individual Tip (electrode Tip) and shape

As shown in FIGS. 15-18, the Tip opening diameter of each glass electrode in Tip1-Tip5 of FIG. 15 is 272nm, 340nm, 298nm, 288nm and 310nm, and the Tip length of the glass electrode is selected to be 8 mm.

Reproducibility of SLMS was performed using glass electrodes with similar tip size and shape. Fig. 15 is a scanning electron microscope image of the geometry of the SLMS glass electrode, 5 selected glass pipette tips. Scale bar: 200 μm. Fig. 16SLMS measures the reproducibility of lysine (LYS, relative standard deviation of 7.3%), histidine (HIS, relative standard deviation of 8.1%), and arginine (ARG, relative standard deviation of 7.5%) in fig. 17.

Repeated measurements were made at levels of 5ppm (34. mu.M) LYS, 5ppm (32. mu.M) HIS and 5ppm (28. mu.M) ARG added to the artificial simulated lysosome fluid (ALF). ALF contains 145mM NaCl, 5mM KCl, 1mM MgCl₂、2mM CaCl₂10mM HEPES, 10mM MES and 10mM glucose (pH adjusted to 4.6 with NaOH, osmolality adjusted to 296mOsm/kg with sucrose). All data were normalized to Tip1 set.

By adopting the electrode tip structure, the spray velocity of pL/min can be obtained, the repeatability and the sensitivity of the detection on substances are higher, and the detection limits on Lysine (LYS), Histidine (HIS) and Arginine (ARG) are 0.045 mu M, 0.042 mu M and 0.038 mu M respectively.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

SEQUENCE LISTING

<110> university of science and technology in China

<120> single cell mass spectrum detection method and detection device

<130> 2021.04.26

<160> 7

<170> PatentIn version 3.3

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Claims (10)

1. A single organelle mass spectrometry detection method, characterized in that the method comprises a method for obtaining single organelle information by combining single organelle patch clamp technology with mass spectrometry technology. 2. single organelle mass spectrometry detection method according to claim 1, is characterized in that, comprises the following steps: Step 1. Use the single organelle patch clamp technique to obtain the contents of the single organelle; Step 2, performing mass spectrometry detection on the content in the single organelle obtained in step 1. 3 . The single organelle mass spectrometry detection method according to claim 1 or 2 , wherein the electrode tip length of the single organelle patch clamp technique used is 5-10 mm, and the tip opening diameter is 0.1-2 μm. 4 . 4 . The single organelle mass spectrometry detection method according to claim 2 , wherein the single organelle patch clamp technique in step 1 comprises the following steps: by applying negative pressure, the contents in the single organelle are sucked into the electrode to remove the Obtain the contents of single organelles. 5. The single organelle mass spectrometry detection method according to claim 2, wherein the single organelle patch clamp technique of the step 1 comprises the following steps: Step 1) culturing cells; Step 2) obtaining the electrophysiological information of the single organelle of the cultured cells in step 1); Step 3) Extract the contents of the single organelle. 6 . The single organelle mass spectrometry detection method according to claim 5 , wherein, in step 2), before the organelle is treated with an electrode, the organelle enlargement treatment is performed. 7 . 7 . The single organelle mass spectrometry detection method according to claim 6 , wherein the composition of the liquid in the electrode used to obtain the electrophysiological information comprises NH ₄ Cl. 8 . The single organelle mass spectrometry detection method according to claim 1, wherein the organelles are derived from the following cells: including HEK-293T cells, mouse embryonic fibroblasts, mouse lung fibroblasts, and bladder cancer cells , Human bladder epithelial immortalized cells, BxPC-3 cells, mouse cardiomyocytes, cardiac fibroblasts, mouse cerebral cortex neurons, glial cells, mouse peritoneal macrophages, mouse embryonic fibroblasts, small At least one of murine lymphofibroblasts. 9 . The single organelle mass spectrometry detection method according to claim 1 , wherein the single organelle comprises at least one of a single lysosome and a single endosome. 10 . 10. A single organelle mass spectrometer detection device, comprising a patch clamp and a mass spectrometer, characterized in that the patch clamp is used to extract the content in the single organelle, and the mass spectrometer is used to analyze the acquired single organelle. The contents were detected by mass spectrometry.

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