CN114518425A - Analytical method for simultaneous detection of metabolites of the cytoplasm and cell membrane of a single immune cell - Google Patents

Analytical method for simultaneous detection of metabolites of the cytoplasm and cell membrane of a single immune cell Download PDF

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CN114518425A
CN114518425A CN202210128161.3A CN202210128161A CN114518425A CN 114518425 A CN114518425 A CN 114518425A CN 202210128161 A CN202210128161 A CN 202210128161A CN 114518425 A CN114518425 A CN 114518425A
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黄光明
侯壮豪
魏海明
郑小虎
陈莹
詹柳娟
庄美慧
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Abstract

The invention discloses an analysis method for simultaneously detecting metabolites of cytoplasm and cell membrane of single immune cell, which comprises the following steps: (1) performing density gradient centrifugation on a sample to be detected to obtain a cell suspension containing immune cells; (2) carrying out immunomagnetic bead screening on the cell suspension by using an immunomagnetic bead antibody to obtain the cell suspension of a single type of immune cells; (3) placing the immune cell suspension under a microscope, adopting a sampling needle to perform single cell sampling, and using a two-stage extraction method of continuously absorbing cytoplasm with low negative pressure and absorbing cell membrane with high pulse negative pressure in the sampling process to improve the success rate of simultaneously detecting the cytoplasm and the metabolite of the cell membrane; (4) placing the sampling needle on an ionization device to ionize the cytoplasm of the single immune cell and the metabolite of the cell membrane; (5) and (3) detecting the ionization information of the metabolites of the cytoplasm and the cell membrane of the single immune cell by using a mass spectrometer to obtain a mass spectrogram of the metabolites of the cytoplasm and the cell membrane of the single immune cell.

Description

Analytical method for simultaneous detection of metabolites of the cytoplasm and cell membrane of a single immune cell
Technical Field
The invention relates to the field of biological mass spectrometry, in particular to an analysis method for simultaneously detecting metabolites of cytoplasm and cell membrane of a single immune cell.
Background
Immune cells can participate in immune response and related life activities, and the immune cells comprise lymphocytes, mast cells, granulocytes, macrophages and the like, wherein the lymphocytes can perform antigen recognition and generate specific immune response, are basic components of an immune system, are widely distributed in cells and are mainly divided into four types of T lymphocytes, B lymphocytes, K lymphocytes and NK lymphocytes. The number of NK cells is small, the ratio of the NK cells to the total number of lymphocytes in peripheral blood and spleen viscera is low, target cells can be directly killed and killed in a non-specific manner, antigen sensitization and antibody participation in advance are not needed, and MHC (major histocompatibility complex) limitation is avoided. The target cells are mainly tumor cells, virus infected cells, larger pathogens and the like. The research on the immune function of immune cells has important biological significance for the diagnosis and treatment of diseases.
In the past, the metabolites of immune cells are usually researched by adopting a cell homogenate mode, the cells are enriched and then crushed, and after further purification, conventional mass spectrometry is carried out, so that the cell specificity analysis aiming at a single cell layer cannot be carried out, and the number of the cells is usually large. For human disease samples with important biological and medical significance, the number of immune cells is often small, and the purified number cannot meet the sampling requirement of conventional chromatographic mass spectrometry.
In the process of disease occurrence, whether the function of immune cells is normally exerted or not needs to be analyzed aiming at a small amount of immune cells at a focus part, and currently, common analysis means such as fluorescence analysis can only research targeted proteins of single cells, cannot analyze most non-targeted proteins, and cannot efficiently screen functional proteins related to diseases at a cell level. And by adopting a scanning electron microscope, a transmission electron microscope and other modes, the morphology of the cell can only be observed, some potential disease-related phenomena are found from the deformation of the cell shape and the subcellular structure, and the deep research on the molecular mechanism of the morphology change is difficult to carry out.
The analysis of the metabolites of the single immune cells at the single cell level is expected to play an important role in guiding the treatment of diseases, the inhibition of tumors, the reconstruction of immune response of mammalian cells and the like, and is an important supplement to technologies such as cell-targeted protein analysis, morphological analysis and the like.
Disclosure of Invention
In order to analyze the metabolites of single immune cells, the invention provides the following technical scheme:
in one aspect, the present invention provides an assay for simultaneously detecting metabolites of the cytoplasm and cell membrane of a single immune cell, comprising the steps of:
step (1), performing density gradient centrifugation on a sample to be detected to obtain a cell suspension containing immune cells;
step (2), carrying out immunomagnetic bead screening on the cell suspension by using an immunomagnetic bead antibody to obtain the cell suspension of a single type of immune cells;
step (3), placing the immune cell suspension under a microscope, adopting a sampling needle to perform single cell sampling, and using a two-stage extraction method of continuously absorbing cytoplasm with low negative pressure and absorbing cell membrane with high pulse negative pressure in the sampling process to improve the success rate of simultaneously detecting the cytoplasm and the metabolite of the cell membrane;
step (4), placing the sampling needle on an ionization device to ionize the cytoplasm of the single immune cell and the metabolite of the cell membrane;
and (5) detecting the ionization information of the metabolites of the cytoplasm and the cell membrane of the single immune cell by using a mass spectrometer to obtain a mass spectrogram of the metabolites of the cytoplasm and the cell membrane of the single immune cell.
In some embodiments, the sample to be tested is selected from blood and tissue.
In some embodiments, the tissue is selected from healthy tissue and diseased tissue, such as cancerous tissue.
In some embodiments, when the sample to be tested is a tissue, the tissue is subjected to enzymatic hydrolysis and then to density gradient centrifugation.
In some embodiments, the density gradient centrifugation medium includes, but is not limited to, sucrose, polysucrose, cesium chloride.
In some embodiments, the density gradient centrifugation medium is selected from sucrose, polysucrose, cesium chloride.
In some embodiments, the immune cell is selected from the group consisting of lymphocytes (e.g., NK cells, T cells, B cells), dendritic cells, monocytes/macrophages, granulocytes, and mast cells.
In some embodiments, the sampling needle is perfused with an electrode fluid selected from deionized water, distilled water, ammonium acetate, ammonium formate, ammonium bicarbonate, ammonium chloride, physiological saline, PBS, and glucose solution.
In some embodiments, a single cell suspension of immune cells is centrifuged, lysate is added to extract the metabolites of the cytoplasmic and cellular membranes, and the cell lysate is then perfused into a sampling needle for analysis.
In some embodiments, the lysing solution is selected from water, methanol, and acetonitrile.
In some embodiments, the sampling needle opening diameter is 1-2.5 microns.
In some embodiments, the sustained low negative pressure is 0.1 to 30kPa for >1 s.
In some embodiments, the pulsed high negative pressure is from 31kPa to 1000kPa for 0.01 to 1 s.
In some embodiments, before the sampling needle is placed on the ionization device, the method further comprises the step of inserting the tip of the sampling needle into deionized water, immersing for 1-5 seconds, and washing away the single cell buffer solution adhered to the surface of the tip.
In some embodiments, in order to prevent the solution at the needle tip from being evaporated and dried to block, the sampling needle should be placed on the single-cell sampling needle ionization device as soon as possible, and the preferred time for placing is within 60 seconds.
In some embodiments, the ionization is performed by placing the sampling needle tip at a distance of 2-10 mm from the entrance of the mass spectrometer.
In some embodiments, the sampling acquisition of the mass spectrometer is started prior to the ionization operation to ensure that signals of metabolites of the cytoplasm and cell membrane of the single cell are obtained.
In some embodiments, the metabolites of the cytosolic and cellular membranes include, but are not limited to, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lactoceramide.
In some embodiments, the metabolites of the cytoplasmic and cellular membranes are selected from the group consisting of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lactosylceramide.
In another aspect, the present invention provides a method for screening a small molecule inhibitor, comprising the steps of:
(1) adding a candidate small molecule inhibitor into the immune cell suspension;
(2) and (3) measuring the signal intensity of the to-be-inhibited substance after the candidate small molecule inhibitor treats the immune cells according to the method, and analyzing the inhibition effect of the candidate small molecule inhibitor on the to-be-inhibited substance, thereby screening the small molecule inhibitor.
In some embodiments, the inhibitor is selected from the group consisting of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lactoceramide.
In some embodiments, the candidate small molecule inhibitor is phospholipase C inhibitor D609.
The present invention can also be used for screening of cell agonists such as drugs, enzyme agonists, hormones, etc., which are capable of enhancing cell functions and promoting biochemical reactions in cells. The invention can also select different surface antibodies to realize the simultaneous mass spectrometry analysis of cytoplasm and cell membrane metabolites of cells such as circulating tumor cells, cancer cells and the like.
In some embodiments, the sustained low negative pressure is a negative pressure of 0.1 to 30kPa for a duration of more than (and including) 1 second for achieving cytoplasmic pumping within NK cells.
In some embodiments, the sustained low negative pressure is achieved using, but not limited to, a 1mL syringe.
In some embodiments, the continuous low negative pressure is a negative pressure of 31kPa to 1000kPa for a duration of 0.01 to 1s seconds, for achieving cell membrane extraction of NK cells, using a mechanical vacuum pump.
In some embodiments, D609, collectively known as the potassium salt of O- (octahydro-4, 7-methano-1H-inden-5-yl) dithiocarbonate, is a selective phosphatidylcholine-specific phospholipase C inhibitor; has the chemical formula of11H15KOS2(ii) a The structural formula is as follows:
Figure BDA0003501413980000041
advantageous effects
Compared with the prior art, the invention has the following beneficial effects:
1. the invention adopts a mode of combining immune sorting and single cell mass spectrum to realize mass spectrum analysis for simultaneously detecting metabolites of single immune cell cytoplasm and cell membrane.
2. Because lipid exists in cell membranes and is easy to fall off in the sampling process, the invention adopts a two-stage extraction method of continuously absorbing cytoplasm under low negative pressure and absorbing cell membranes under high pulse negative pressure, thereby obviously improving the success rate of simultaneously detecting the cytoplasm and metabolites of the cell membranes.
3. The invention provides an immunomagnetic bead-microsampling-mass spectrometry combined analysis method, which selects specific antibodies, screens immune cells with high selectivity, and distinguishes various immune cell types, thereby carrying out single-cell mass spectrometry on single immune cells.
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FIG. 1 shows a flow chart of an assay method of the present invention for simultaneous detection of metabolites of the cytoplasm and cell membrane of mammalian single immune cells.
FIG. 2 shows a schematic diagram of an analytical method combining immunomagnetic bead-microsampling-mass spectrometry for simultaneous detection of metabolites of the cytoplasm and cell membrane of mammalian single immune cells, including cell dispersion; sorting immunomagnetic beads; microsampling of the cytoplasm and cell membranes; ionizing metabolites of the cytoplasm and cell membrane; metabolite signals of both cytosol and cell membranes are acquired simultaneously.
FIG. 3 shows a schematic of single cell microsampling, including a needle tip reaching the cell; the needle tip aspirates the cell cytosol and the metabolites of the cell membrane.
FIG. 4 shows flow sorting of NK cells (CD 3)-CD16+CD56+) The purity effect diagram of (1).
FIG. 5 shows mass spectra of seven sphingomyelins in peripheral blood, liver and liver cancer tissue mono-immune NK cells obtained by analysis by the method of the present invention.
FIG. 6 shows mass spectra of lipids such as lecithin in peripheral blood, liver and liver cancer tissue mono-immune NK cells analyzed by the method of the present invention.
FIG. 7 shows a statistical graph of the signal levels of seven sphingomyelins in peripheral blood, liver and liver cancer tissue mono-immune NK cells obtained by analysis by the method of the present invention. Wherein the abscissa is the type of sphingomyelin and the ordinate is the signal intensity.
FIG. 8 shows a statistical graph of signal levels of metabolites associated with the upstream pathway of sphingomyelin synthesis (serine/serine, dihydrosphingosine/sphingosine, dihydroceramide/dihydroceramide, sphingosine/sphingosine, acetylcholine/acetylcholine, choline/choline, phosphocholine/phorylcholine) in peripheral blood, liver and liver cancer tissue mono-immune NK cells analyzed by the method of the present invention.
Figure 9 shows the synthetic pathway for sphingomyelin.
FIG. 10 shows secondary mass spectral fragment signatures of six sphingomyelins obtained by analysis according to the method of the present invention.
FIG. 11 shows the linear range of sphingomyelin obtained by single immune cell mass spectrometry and conventional liquid phase mass spectrometry of the invention.
Figure 12 shows sphingomyelin mass spectra of mono-immune NK cells obtained by the method of the invention after action with 0, 25, 100, and 400uM of D609 inhibitor.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
Example 1 analysis method for simultaneous detection of metabolites of the cytoplasm and cell membrane of a single immune cell in combination with immunomagnetic bead-microsampling-mass spectrometry
The analysis method for simultaneously detecting the metabolites of the cytoplasm and the cell membrane of the single immune cell by combining the immunomagnetic beads with the microsampling and mass spectrometry comprises the following steps:
step 1, performing density gradient centrifugation on human fresh peripheral blood by using sucrose as a centrifugation medium, wherein the density of monocytes is different from that of lymphocytes, the monocytes can be separated after the gradient centrifugation, the lymphocytes are separated from supernatant by centrifugation, and the supernatant is taken and contains a single cell suspension of the lymphocytes; fresh normal tissues or cancer tissues approved by ethical committee of human beings are cut into small fragments, then the small fragments are placed in PBS or NK cell complete culture medium (CC 000) for 0.25 percent of trypsinization and digestion for 30 minutes of intercellular substance, and a single cell suspension of lymphocytes is obtained by adopting a gradient density centrifugation method;
step 2, adding immunomagnetic bead antibodies into lymphocyte suspensions obtained from peripheral blood, normal tissues and cancer tissues, and performing high-specificity sorting of NK cells by using a magnetic immunomagnetic bead sorting column (Meitian and whirlwind, mini-Macs) to obtain targeted immune cell single cell suspensions with purity of more than 90% (as shown in FIG. 4); and resuspending the NK cells in a single cell phosphate buffered saline (PBS buffer);
step 3, dripping the single cell suspension sample in a culture dish, carrying out micro-area positioning under a microscope, placing a needle point of a borosilicate sampling needle (setter B100-50-10) under the microscope to confirm the opening size of the sampling needle, wherein the diameter of the opening of the needle point is 1-2.5 microns, filling an electrode liquid (185mM ammonium acetate and 80mM ammonium bicarbonate mixed liquid) in the sampling needle, fixing the needle on a micro-operation platform and connecting the needle with an air pump, and carrying out single immune cell sampling; in the process of sucking NK cells by the sampling needle for sampling and in the process of moving out the single cell suspension after successful sampling, keeping the continuous low negative pressure of an air pump to be kept for holding (0.1-30kPa, >1s), sucking cytoplasm, then pulsing high negative pressure to suck cell membranes (31kPa-1000kPa, 0.01-1s), sucking the NK cell membranes at the needle point of the sampling needle (as shown in figure 3), taking out the sampling needle, inserting the needle point into deionized water, immersing for 3 seconds, and washing away single cell phosphate buffer solution (PBS buffer solution) adhered to the surface of the needle point to reduce ion inhibition;
step 4, placing the sampling needle on a single NK cell sampling needle ionization device, adopting an induction type electrode to avoid the sampling needle from being polluted to carry out single cell ionization, adjusting the output waveform to be sine wave, adjusting the voltage frequency to be 1000Hz, adjusting the voltage Vpp to be 3kV, enabling the needle tip to be 2-10 mm away from the mass spectrum inlet, easily breaking the needle tip when the needle tip is too close to the mass spectrum inlet, and enabling the needle tip to be too far away from the ionization device, so that the ionization effect is not good;
step 5, the orbitrap mass spectrum is an exact Plus mass spectrometer (ThermoFisher Scientific, CA, USA), and the acquisition parameters are as follows: the mass scanning range is 60-900%, the mass resolution is 140000, the AGC threshold value is 1e6, the positive ion mode is adopted, the S-Lens radio frequency level is 50%, the capillary tube transmission temperature is 275 ℃, the average spectrum frame number is 1 frame, the acquisition time is 5min, the ionization phase is started first, the ionization information of the metabolites of the single cell can be ensured to be completely acquired by the mass spectrum, and the information loss can not be caused;
step 6, in the analysis of single NK cells, the present invention detected up to 42 lipid components, including seven sphingomyelins (SM (d34:1), SM (d36:2), SM (d38:0), SM (d36:1), SM (d36:0), SM (d32:2), SM (d41:2), fig. 5), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), and lactosylceramide (La) (fig. 6). 7 different SM molecules were detected in a single NK cell (fig. 5). Single immune cell analysis of NK cells from different sources showed a significant reduction of the overall sphingomyelin levels of NK cells within the tumor compared to the sphingomyelin content of hepatic and peripheral NK cells. Specifically, the levels of 5 SM molecules (SM (d34:1) (d34:1 indicates the presence of an alkyl chain containing 34 carbon atoms and 1 unsaturated double bond), SM (d38:0) (d38:0 indicates the presence of an alkyl chain containing 38 carbon atoms and 0 unsaturated double bond), SM (d36:1) (d36:1 indicates the presence of an alkyl chain containing 36 carbon atoms and 1 unsaturated double bond), SM (d36:0) (d36:0 indicates the presence of an alkyl chain containing 36 carbon atoms and 0 unsaturated double bond) and SM (d32:2) (d32:2 indicates the presence of an alkyl chain containing 32 carbon atoms and 2 unsaturated double bonds) in intratumoral NK cells were significantly reduced (p <0.05 compared to hepatic NK cells and peripheral NK cells) (FIG. 7.) serine metabolism in cancer was deregulated, while serine was a precursor metabolite of sphingomyelin biosynthesis from the head, respectively, NK cells co-cultured with cancer cells showed significantly reduced serine levels (both extracellular and intracellular levels) compared to NK cells cultured alone (fig. 8 and 9), with serine detection by cells as intracellular serine levels and serine detection in the medium as extracellular levels. In addition, sphingomyelin synthesis generally proceeds via two pathways, one of which is associated with ceramide synthesis (e.g., sphingosine, dihydroceramide, and dihydrosphingosine) and the other of which is associated with lecithin synthesis (e.g., acetylcholine, choline, and phosphorylcholine), which ultimately synthesize sphingomyelin under the action of enzymes, as shown in fig. 9. Single cell detection of the related synthetic metabolites of both pathways found a significant reduction of other metabolites associated with SM biosynthesis (e.g., sphingosine, dihydroceramide and sphinganine) in NK cells co-cultured with cancer cells (p <0.05, fig. 8 and 9). Phosphatidylcholine (PC) can also be used as a precursor for SM biosynthesis, with no change in intratumoral NK cell levels compared to phosphatidylcholine or related upstream metabolites (e.g., acetylcholine, choline, and phosphorylcholine) in hepatic NK cells or peripheral NK cells (FIGS. 8 and 9). Together, these results suggest that the observed reduction in NK cell SM levels within tumors appears to be caused by a dysregulation of serine metabolism associated with certain cancer cells.
Example 2 Secondary Mass Spectrometry characterization of sphingomyelin
According to the procedures of step 1 and step 2 of example 1, a suspension of the mono-immune NK cells was obtained, 100 μ l of pure methanol was added thereto to perform cell lysis, and a cell lysate was obtained and injected into a sample injector.
Step 3, the ion trap mass spectrum is an LTQ mass spectrometer (ThermoFisher Scientific, CA, USA), and the acquisition parameters are: the mass scanning range is 60-900, the positive ion mode is realized, the radio frequency level of S-Lens is 60%, the capillary transmission temperature is 275 ℃, the average spectrum frame number is 1 frame, the collision fragmentation energy is 35eV, the mass range of the selected parent ions is 1.0Da, the LTQ mass spectrometer is used for tandem mass spectrometry, and the tandem mass spectrometry is a detection mode equipped in the LTQ mass spectrometer;
step 4, performing confirmatory analysis on the NK cell lysate by using tandem mass spectrometry (MS/MS), selecting typical 6 sphingomyelin analytes for molecular fragment analysis, and obtaining a characteristic daughter ion [ SM (d34:1) -O + H ] related to the structure through secondary fragment analysis, wherein the characteristic daughter ion is sphingomyelin SM (d34:1) parent ion 703.6 shown in figure 10]+685.6/[SM(d34:1)-C3H9N+H]+644.5/[SM(d34:1)-C16H30O+H]+463.3, confirming the presence of sphingomyelin SM (d34:1) in the sample; sphingomyelin SM (d38:0) parent ion 761.6, which, by secondary fragment analysis, yields the structurally related characteristic ion [ SM (d38:0) -C3H9O+H]+702.6/[SM(d38:0)-C5H14NO4P+H]+578.6, confirming the presence of sphingomyelin SM (d38:0) in the sample; sphingomyelin SM (d36:1) parent ion 769.6, which, by secondary fragment analysis, yields the structurally related characteristic daughter ion [ SM (d36:1) -C3H9N+K]+710.5/[SM(d36:1)-C5H12N+H]+645.5/[SM(d36:1)-C5H14NO4P+K]+586.5/[SM(d36:1)-C18H34O+H]+465.3, confirming the presence of sphingomyelin SM (d36: 1); sphingomyelin SM (d36:0) parent ion 771.6, obtained by secondary fragment analysisStructure-related characteristic daughter ions [ SM (d36:0) -C3H9N+K]+712.6/[SM(d36:0)-C5H12N+H]+647.6/[SM(d36:0)-C5H14NO4P+K]+588.6, confirmation of the presence of sphingomyelin SM (d36:0) in the sample; sphingomyelin SM (d42:2) parent ion 813.6.6, which, by secondary fragment analysis, yields the structurally related characteristic daughter ion [ SM (d42:2) -C3H9N+H]+754.6/[SM(d42:2)-H2O+H]+795.7/[SM(d42:2)-C5H14NO4P+H]+630.6, confirming the presence of sphingomyelin SM (d42:2) in the sample; sphingomyelin SM (d43:2) parent ion 837.6, which, by secondary fragment analysis, yields the structurally related characteristic daughter ion [ SM (d43:2) -C3H9N+K]+778.6/[SM(d43:2)-C5H13NO4P+H]+654.6, confirming the presence of sphingomyelin SM (d43:2) in the sample. This result is consistent with the information for sphingomyelin detected in single cells from example 1. It was confirmed that the sphingomyelin fraction measured in example 1 was correct.
Example 3 Single immune cell Mass Spectrometry and liquid chromatography Mass Spectrometry quantitative analysis of sphingomyelin
Step 1, preparing 0.2, 1, 5, 20 and 50mg/L standard solutions of sphingomyelin SM (d34:1) and SM (d36:1) respectively;
fixing a sampling needle on a micro-operation platform and sucking a sphingomyelin standard solution in a single cell sampling mode;
step 3, placing the sampling needle on a single-cell sampling needle ionization device, performing single-cell ionization by adopting an induction type electrode, adjusting the output waveform to be sine wave, adjusting the voltage frequency to be 1000Hz, adjusting the voltage Vpp to be 3kV, and enabling the needle tip to be 2 mm away from a mass spectrum inlet;
step 4, the orbitrap mass spectrum is an exact Plus mass spectrometer (ThermoFisher Scientific, CA, USA), and the acquisition parameters are as follows: the mass scanning range is 60-900, the mass resolution is 140000, the AGC threshold value is 1e6, the positive ion mode is adopted, the S-Lens radio frequency level is 50%, the capillary transmission temperature is 275 ℃, the average spectrum frame number is 1 frame, the acquisition time is 5min, the method is started before the ionization stage, and the starting before the ionization stage can ensure that all the metabolite ionization information of the single cell is acquired by mass spectrometry, so that the information loss can not be caused;
step 5, pouring the sphingomyelin solution into a chromatographic sample injection bottle, and placing the chromatographic sample injection bottle into an automatic sample injector;
step 6, parameters of chromatograph-mass spectrometer TripleTOF 5600+ (AB SCIEX, America) were set as follows, C18 analytical column, mobile phase a containing 10mM ammonium acetate and 0.1% aqueous formic acid, mobile phase B containing 10mM ammonium acetate and 0.1% methanol: isopropyl alcohol: water (60:40:1), gradient elution procedure: 0-2min, 35-80% of mobile phase B; 2-7min, 80-100% of mobile phase B; 7-17min, 100% mobile phase B; the chromatograph-mass spectrometer is used for a reference testing instrument, and the orbital trap mass spectrometer is used for testing the method provided by the invention.
This method carried out a standard benchmark test and we evaluated the analytical performance of the method of the invention with LC-MS (fig. 11) in detecting reference standard sphingomyelins (SM (d34:1) and SM (d36:1)) at concentrations ranging from 0.2 to 50 mg/L. The slope of the SM (d34:1) standard curve was 2.021X 10 using the single immunocytological method-2，R20.9925, RSD 7.6%, and the slope of the SM (d36:1) standard curve is 2.012 × 10-2，R20.9977, RSD 7.3%. The slope of the SM (d34:1) standard curve was 1.977X 10 using liquid chromatography-mass spectrometry-2，R20.9981, RSD 4.0%, the slope of the SM (d36:1) standard curve is 1.979 × 10-2，R20.9923, RSD 3.3%. Illustrating that the method is comparable to the linear range and sensitivity obtained by conventional standard chromatographic mass spectrometry in the analysis of standard solutions of sphingomyelin. The method and the standard chromatographic mass spectrometry can achieve the same analysis effect in the analysis of the standard substance. However, according to example 1, the present invention is advantageous in that sphingomyelin analysis of individual cells can be achieved. Whereas liquid chromatography can only achieve sphingomyelin analysis of multicellular lysates.
Example 4 lipid Signal Change of immune cells after treatment with Small molecule inhibitors of sphingomyelin synthase (D609)
Referring to step 1 and step 2 of example 1, the obtained NK cells were cultured in NK cell complete medium containing interleukin-15 (IL-15) of 5ng/ml and interleukin-2 (IL-2) of 100U/ml for activating NK cells, and D609(MedChemExpress, 83373-60-8) containing 25, 100, 400. mu.M/L or phosphate buffer solution was directly added to the cell supernatant for 24 hours.
Referring to step 3, step 4 and step 5 of example 1, sphingomyelin signals of single immune cells were obtained and it was found that small molecule inhibitors of sphingomyelin synthase (D609) decreased SM levels in peripheral blood NK cells (normal donors) (fig. 12). The bold line in fig. 12 indicates 7 types of sphingomyelin, the ordinate represents signal intensity, the content of sphingomyelin in the cells after D609 treatment tends to decrease as a whole, the decrease of sphingomyelin increases with increasing drug concentration, and the signal intensity of sphingomyelin is lowest when the concentration of D609 reaches 400 μ M. D609 shows that the composition has the effect of inhibiting NK cell sphingomyelin synthesis. NK cells are hindered in sphingomyelin synthase, which results in decreased sphingomyelin content of the cells.
The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only examples of the present invention, and should not be construed as limiting the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. An assay for simultaneously detecting metabolites of the cytoplasm and cell membrane of a single immune cell comprising the steps of:
step (1), performing density gradient centrifugation on a sample to be detected to obtain a cell suspension containing immune cells;
step (2), carrying out immunomagnetic bead screening on the cell suspension by using an immunomagnetic bead antibody to obtain the cell suspension of a single type of immune cells;
step (3), placing the immune cell suspension under a microscope, adopting a sampling needle to perform single cell sampling, and using a two-stage extraction method of continuously absorbing cytoplasm with low negative pressure and absorbing cell membrane with high pulse negative pressure in the sampling process to improve the success rate of simultaneously detecting the cytoplasm and the metabolite of the cell membrane;
step (4), placing the sampling needle on an ionization device to ionize the cytoplasm of the single immune cell and the metabolite of the cell membrane;
and (5) detecting the ionization information of the metabolites of the cytoplasm and the cell membrane of the single immune cell by using a mass spectrometer to obtain a mass spectrogram of the metabolites of the cytoplasm and the cell membrane of the single immune cell.
2. The analytical method for the simultaneous detection of metabolites of the cytoplasmic and cellular membranes of a single immune cell according to claim 1, wherein the sample to be tested is selected from the group consisting of blood and tissue, preferably the tissue is selected from the group consisting of healthy tissue and diseased tissue, such as cancerous tissue.
3. The method according to claim 2, wherein the sample to be tested is a tissue, and the tissue is subjected to enzymolysis and then to density gradient centrifugation.
4. The assay for the simultaneous detection of metabolites of the cytoplasm and cell membrane of a monoimmune cell according to any one of claims 1 to 3, wherein said immune cell is selected from the group consisting of lymphocytes (such as NK cells, T cells, B cells), dendritic cells, monocytes/macrophages, granulocytes and mast cells.
5. The assay for simultaneous detection of metabolites of the cytoplasm and cell membrane of a single immune cell according to any one of claims 1-4, wherein said sampling needle is perfused with an intra-electrode fluid selected from the group consisting of deionized water, distilled water, ammonium acetate, ammonium formate, ammonium bicarbonate, ammonium chloride, physiological saline, PBS, glucose solution and any mixture thereof.
6. The method according to any one of claims 1 to 5, wherein the opening of the sampling needle has a diameter of 1 to 2.5 μm.
7. The assay for the simultaneous detection of metabolites of the cytoplasm and cell membrane of a single immune cell according to any one of claims 1-6, wherein said continuous low negative pressure is 0.1-30kPa for >1s, optionally said pulsed high negative pressure is 31kPa-1000kPa for 0.01-1 s.
8. The analytical method for simultaneously detecting metabolites of the cytoplasm and the cell membrane of a single immune cell according to any one of claims 1 to 7, further comprising the step of immersing the tip of the sampling needle in deionized water for 1 to 5 seconds to wash away the buffer solution of the single cell adhered to the surface of the tip before placing the sampling needle on the ionization device.
9. The method according to any one of claims 1 to 8, wherein the ionization is carried out by placing the tip of the sampling needle at a distance of 2 to 10mm from the entrance of the mass spectrometer.
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