CN111397985B - Single cell mass spectrometry - Google Patents
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- CN111397985B CN111397985B CN202010288305.2A CN202010288305A CN111397985B CN 111397985 B CN111397985 B CN 111397985B CN 202010288305 A CN202010288305 A CN 202010288305A CN 111397985 B CN111397985 B CN 111397985B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0431—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for liquid samples
- H01J49/0445—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for liquid samples with means for introducing as a spray, a jet or an aerosol
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Abstract
The invention provides a single cell mass spectrometry method, which comprises the following steps: carrying out cell fixation and dilution treatment on cells to be detected so as to obtain a pretreated cell suspension; injecting the pretreated cell suspension into an inner cavity from the tail end of the capillary, wherein an electrode is inserted into the inner cavity; applying a voltage to the electrodes such that the cells migrate to the capillary tip; completely evaporating the liquid in the capillary; and dropping auxiliary liquid into the tip of the capillary to make the auxiliary liquid enter the capillary to contact with cells to extract a target object, and simultaneously applying voltage to the electrode to perform electrospray so as to ionize the auxiliary liquid containing the target object, spraying the auxiliary liquid out of the tip, and entering a sample inlet of a mass spectrometer to perform mass spectrometry. The method can realize the sampling and mass spectrometry of single cells without depending on a high-precision control platform, can realize the identification of the position of the double bond of unsaturated lipid in the single cells by combining the photochemical derivation process, and has good application prospect.
Description
Technical Field
The present invention relates to the field of biology. In particular, the invention relates to a single cell mass spectrometry method.
Background
There can be large differences, i.e., cellular heterogeneity, between individuals in a population of cells of the same genotype. The cells have obvious difference in all aspects of gene transcription, protein expression, metabolism and the like. Therefore, single cell analysis is of great significance for studying intercellular heterogeneity. A variety of techniques have been widely used for single cell analysis, such as single cell sequencing, fluorescence detection techniques, microfluidic techniques, flow cytometry, mass spectrometry, and the like. Among them, mass spectrometers are widely used in single cell analysis due to their characteristics of high sensitivity, accurate structural identification, quantitative analysis, etc. However, the small size and small volume of the cells themselves pose a significant challenge to single cell mass spectrometry. For example, the diameter of a single cell is 1 to 30 μm, and the volume is of the order of fL to nL. Excessive dilution of metabolites in cells can result in too low concentration, which exceeds the mass spectrum detection range; if the dilution is too small, the amount of the sample available for analysis is too small, and the difficulty in manipulating the sample is greatly increased.
Currently, a variety of single cell mass spectrometry techniques have been developed, such as living cell mass spectrometry, capillary micro-sampling mass spectrometry, probe mass spectrometry, and the like. The technologies mostly depend on a high-precision operation platform, capillary glass tubes, metal probes and the like are used for penetrating into cells to absorb/extract metabolites in the cells, and therefore single cell control, sampling and analysis are achieved. Another class of techniques relies on desorption techniques such as matrix-assisted laser desorption ionization, desorption electrospray ionization, nanoarray laser desorption ionization, and the like. These techniques also need to rely on high precision operating platforms to achieve sampling of individual cells. Therefore, the sampling cost is greatly improved, the convenience of operation is reduced, and the popularization and the application are not facilitated.
Therefore, methods for single cell mass spectrometry are still under investigation.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a single-cell mass spectrometry method, which can realize the sampling of single cells without depending on a high-precision control platform, is particularly suitable for lipid compounds with carbon-carbon double bonds, realizes the identification and analysis of isomers of the carbon-carbon double bonds of single-cell lactones for the first time, and has important significance on the research of the metabolism of the lactones in organisms. Moreover, the method is simple and convenient to operate, low in cost and suitable for large-scale application.
The invention provides a single cell mass spectrometry method. According to an embodiment of the invention, the method comprises: carrying out cell fixation and dilution treatment on cells to be detected so as to obtain a pretreated cell suspension; injecting the pretreated cell suspension into a cavity from the tail end of the capillary, wherein an electrode is inserted into the cavity; applying a voltage to the electrodes such that cells migrate to the capillary tip; allowing the liquid in the capillary to evaporate completely; and dropping auxiliary liquid into the tip of the capillary, enabling the auxiliary liquid to enter the capillary and contact with the cells to extract a target object, and simultaneously applying voltage to the electrode to perform electrospray so as to enable the auxiliary liquid containing the target object to be ionized, to be ejected from the tip, to enter a sample inlet of a mass spectrometer, and to perform mass spectrometry.
In the method according to an embodiment of the present invention, the cell suspension subjected to the cell fixing treatment is diluted by a certain factor so that the number of cells in the pretreated cell suspension added to the capillary approaches that of a single cell. By utilizing the property that the cell surface is negatively charged, when a negative voltage is applied to the electrode, the cell will migrate to the capillary tip. Therefore, the capillary, the metal probe and the like are not used for deeply reaching the inside of the cell to extract the target object, so that a high-precision control platform is not required, the implementation is convenient, and the cost is reduced.
Subsequently, the liquid in the capillary tube is evaporated, so that the subsequent auxiliary liquid can enter the inner cavity of the capillary tube through the capillary action. Because the liquid content in the capillary is very small, complete evaporation of the liquid can be realized in a short time. Then, auxiliary liquid is dripped at the tip of the capillary tube, and the auxiliary liquid enters the inner cavity of the capillary tube under the capillary action and contacts with cells, so that target substances in the cells are dissolved out, and the purpose of extraction is achieved. Meanwhile, voltage is applied to the electrodes to realize electrospray, and the ionized target object is ejected into a mass spectrometer to perform mass spectrometry. Therefore, the single-cell mass spectrometry method provided by the embodiment of the invention is simple and convenient to operate, high in efficiency, low in cost and suitable for large-scale application.
According to an embodiment of the present invention, the single-cell mass spectrometry method may further have the following additional technical features:
according to an embodiment of the present invention, the target is a lipid substance having a carbon-carbon double bond, and the method further comprises the following steps to facilitate identification of the position of the carbon-carbon double bond in the lipid substance: and carrying out photochemical derivatization treatment on the cell suspension subjected to the cell fixing treatment, and then carrying out the dilution treatment on the obtained cell suspension.
According to an embodiment of the invention, the derivatizing agent used in the photochemical derivatizing treatment is selected from the group consisting of water-soluble carbonyl compounds, preferably 2-acetylpyridine, 3-acetylpyridine or 4-acetylpyridine.
According to an embodiment of the invention, the photochemical derivatization treatment comprises: and mixing the cell suspension with the derivatization reagent, and irradiating for 1-15 minutes under 250-260 nm ultraviolet light.
According to an embodiment of the present invention, theThe number of cells in the pretreated cell suspension was 2X 103～1×104And each/mL, wherein the volume of the pretreated cell suspension added into the capillary tube is 0.3-0.6 mu L.
According to an embodiment of the present invention, the cell fixing solution used in the cell fixing treatment is selected from a paraformaldehyde-based chemical, preferably paraformaldehyde or glutaraldehyde.
According to an embodiment of the invention, the outer diameter of the capillary tip is smaller than the diameter of the cells in the pretreated cell suspension.
According to an embodiment of the invention, the outer diameter of the capillary tip is 3-10 μm.
According to an embodiment of the present invention, the auxiliary liquid is selected from at least one of methanol, ethanol, acetonitrile, acetone, chloroform, isopropanol, and water.
According to an embodiment of the invention, the target is selected from at least one of glycerophosphocholine, a diglyceride, a triglyceride, a fatty acid, and cholesterol oleate.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic flow diagram of a single-cell mass spectrometry method according to an embodiment of the present invention;
FIG. 2 shows a diagram of a reaction apparatus according to one embodiment of the present invention;
FIG. 3 is a schematic diagram showing a capillary glass tube structure according to an embodiment of the present invention, wherein (a) is a schematic diagram showing a structure of a capillary glass tube and an electrode, 1 is the capillary glass tube, and 2 is the electrode; (b) microscopic image of single MCF7 cells migrated to the tip of a drawn capillary glass tube;
FIG. 4 shows a lipid mass spectrum of a single MCF7 cell according to one embodiment of the invention;
FIG. 5 shows a lipid mass spectrum of MCF7 cells after a single photochemical derivatization, according to an embodiment of the invention;
FIG. 6 shows a schematic comparison of the conversion of 3 acetylpyridines according to one embodiment of the present invention;
FIG. 7 shows a tandem mass spectrometry spectrum of a carbon-carbon double bond isomer of glycerophosphorylcholine (PC) determined within a single MCF7, according to an embodiment of the invention;
figure 8 shows a tandem mass spectrum for determining the carbon-carbon double bond isomeric structure of Fatty Acids (FA), Diglycerides (DAG), Triglycerides (TAG), cholesterol oleate (CE) within a single MCF7 according to one embodiment of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
For ease of understanding, the single cell mass spectrometry method is described in detail below.
The invention provides a single cell mass spectrometry method. According to an embodiment of the invention, referring to fig. 1, the method comprises:
s100 cell fixation and dilution treatment
In this step, the cell fluid to be tested is subjected to cell fixation and dilution treatment to obtain a pretreated cell suspension.
Through cell fixation, proteins, antigens, cytoskeletons and the like in cells are precipitated or fixed and positioned at original positions in the cells, the reaction of exogenous and endogenous lytic enzymes is stopped or reduced, the tissues are prevented from being subjected to cell autolysis due to prolonged in vitro time, and the dispersion, damage and loss of soluble proteins, fats and sugar substances of the cells are reduced. In particular, cell fixation can be performed using a paraformaldehyde based chemical, particularly paraformaldehyde or glutaraldehyde.
It should be noted that the present invention is not limited to the type of target in the cell suspension, and any cell metabolite can be analyzed by the single-cell mass spectrometry method of the present invention, especially lipid substances having carbon-carbon double bonds, such as at least one of glycerophosphorylcholine, diglyceride, triglyceride, fatty acid, and cholesterol oleate. The isomeric structure of intracellular lipids and the like is relatively complex. For example, in the case of glycerophosphorylcholine, the structural information includes various information such as fatty acid chain composition, fatty acid chain position, unsaturated double bond configuration, and the like. The relative content change of the isomers of the lipid has important significance for the development of organisms.
According to an embodiment of the present invention, the target is a lipid substance having a carbon-carbon double bond, and the method further comprises the following steps to facilitate identification of the position of the carbon-carbon double bond in the lipid substance: and carrying out photochemical derivatization treatment on the cell sap after cell fixation treatment, and then diluting the obtained cell suspension.
The unsaturated lipid substance is subjected to derivatization reaction to obtain an epoxidized product, and the molecular weight of the epoxidized product is increased by a specific value compared with that of the original lipid (namely, the lipid molecules before derivatization). And (3) selecting the epoxidation product of the analyte to be subjected to tandem mass spectrometry, wherein the selection is based on that the mass number of the epoxidation product is increased by a specific value compared with that of the pre-derivatized lipid molecule. In CID fragmentation mode, the derivatized lipid molecule will generate specific diagnostic ions for the C ═ C position, and each pair of diagnostic ions will differ by that particular value, thereby allowing identification of the C ═ C position of the lipid.
According to an embodiment of the invention, the derivatizing agent used for the photochemical derivatizing treatment is selected from the group consisting of water-soluble carbonyl compounds, preferably 2-acetylpyridine, 3-acetylpyridine or 4-acetylpyridine. The inventor finds that the derivatization treatment of single cells has higher requirements on the adopted derivatization reagent, and certain reagents can cause the failure of experimental single cell detection, such as acetone. Acetone is a fat-soluble organic solvent, and is added into a cell suspension containing a plurality of cells to easily cause cell membrane dissolution, so that metabolites in the cells are mixed into a whole, and the requirement of single cell detection cannot be met. Therefore, the inventor finds that among a plurality of reagents, the water-soluble carbonyl compound, particularly 2-acetylpyridine, 3-acetylpyridine or 4-acetylpyridine has better effect, can enable the reaction to be carried out in aqueous solution, does not cause cell rupture, does not cause intercommunication of internal metabolites among cells, can obtain detectable epoxide, has high conversion rate and ensures the accuracy of mass spectrum detection.
According to an embodiment of the invention, the photochemical derivatization treatment comprises: mixing the cell suspension with a derivatization reagent, and irradiating for 1-15 minutes under 250-260 nm ultraviolet light. The light derivatization reaction can be carried out in a quartz reaction cell, the reaction apparatus being shown in FIG. 2. Thus, the epoxidized derivative can be effectively obtained, thereby facilitating subsequent mass spectrometric detection and identification of the C ═ C position.
According to an embodiment of the present invention, the number of cells in the pretreated cell suspension is 2X 103～1×104one/mL. Therefore, the number of cells added into the capillary tube subsequently can be about 1-5, and single cell analysis is realized.
S200, injecting the pretreated cell suspension into the inner cavity from the tail end of the capillary, and inserting the metal electrode into the capillary
In this step, the pretreated cell suspension is injected into the lumen from the end of the capillary, and the metal electrode is inserted into the hair cell.
According to the embodiment of the invention, the capillary glass tube can be drawn by a P1000 pin drawing machine, and the diameter of the tip of the glass tube is 3-10 mu m. 0.3-0.6. mu.L of predicted cell sap (2X 10) can be transferred using a 2.5. mu.L standard pipette3～1×104one/mL) to the tip of the capillary glass tube, statistically, 1-5 cells will be added into the capillary with a high probability. Because the number of cells is small, the capillary containing 1-5 cells can be directly adopted for subsequent experiments. If the requirement for analysis is high, capillaries with an excessive number of cells (e.g., containing 2, 3, 4 or 5 cells) can be discarded and selected to contain only a specific number of cells (e.g., containing only 2, 3, 4 or 5 cells) by microscopic observation1, 2) capillaries were subjected to subsequent experiments.
S300 applying a voltage to the electrodes causes the cells to migrate to the capillary tip
In this step, a voltage is applied to the electrodes, causing the cells to migrate to the capillary tip.
At present, capillary glass tubes, metal probes and the like are mostly used to deeply penetrate into cells, and the high-precision operation platform is required to be relied on for extracting and extracting metabolites in the cells. According to the invention, 0.3-0.6 mu L of pretreated cell suspension with 1-5 cells is added into the capillary from the tail end of the capillary, and the amplitude of the applied voltage is 800-2000V after negative voltage is applied by utilizing the characteristic of negative charge of the cells. The voltage can be applied through the stainless steel electrode wire, and the application time is 5-60 s. Using microscopic observation, it was confirmed that the cells were migrated to the tip of the capillary glass tube, as shown in FIG. 3.
According to an embodiment of the invention, the outer diameter of the capillary tip is smaller than the diameter of the cells in the pre-treated cell suspension. The outer diameter of the capillary tip is smaller than the diameter of the cell, otherwise the cell flows out of the tip, and the isolation and subsequent sampling analysis of the single cell cannot be realized. According to an embodiment of the invention, the capillary tip has an outer diameter of 3 to 10 μm.
S400 complete evaporation of liquid in capillary
In this step, the liquid in the capillary is completely evaporated. And (3) standing the capillary for a period of time (generally 2-5 minutes) to completely evaporate the liquid in the capillary. The liquid in the capillary has small volume and can be completely evaporated in a short time. Observing under a microscope, the point has no liquid, and the evaporation is considered to be finished. The liquid evaporation enables the opening at the front end of the capillary tube to absorb the auxiliary liquid in the subsequent step through the capillary action, thereby realizing the extraction and mass spectrometry of the metabolites in the cells.
S500, auxiliary liquid is dripped to the tip of the capillary tube, the auxiliary liquid enters the capillary tube to be contacted with cells, a target object is extracted, and meanwhile, voltage is applied to an electrode to perform electrospray.
In this step, an auxiliary liquid is dropped onto the tip of the capillary, the auxiliary liquid is caused to enter the capillary and come into contact with the cells, and the target is extracted, and at the same time, a voltage is applied to the electrodes to perform electrospray, so that the auxiliary liquid containing the target is ionized, ejected from the tip, and enters the sample inlet of the mass spectrometer for mass spectrometry. Thereby, mass spectrometry is realized.
The type of the auxiliary liquid is not limited in the present invention, and may be any extraction reagent disclosed in the art, as long as the target substance in the cell can be extracted, and the type of the auxiliary liquid may be flexibly selected according to the actual situation. According to an embodiment of the present invention, the auxiliary liquid includes, but is not limited to, methanol, ethanol, acetonitrile, acetone, isopropanol, chloroform, water, and the like.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1 lipid substance analysis in Single MCF7 cells
Fixation of cells: cultured MCF7 cells (7-8 μm) were transferred to PBS, centrifuged at 1200rpm, resuspended in Phosphate Buffered Saline (PBS) containing 2.5% glutaraldehyde, centrifuged twenty minutes later, and the cells were collected and resuspended in water to give a cell suspension.
Preparing a capillary glass tube: capillary glass tubes with a tip diameter of 5 μm were drawn using a P1000 pin drawing machine.
Mass spectrometry analysis:
(1) add 0.5. mu.L of cell suspension into capillary glass tube from the tail end of capillary glass tube, select capillary glass tube containing 1 cell under microscope, and migrate single MCF7 cell to the front end of capillary glass tube using-1.2 kV voltage.
(2) And waiting for the liquid in the capillary glass tube to evaporate.
(3) A voltage of- +1.8kV was applied while dropping an auxiliary droplet from the tip. The auxiliary drop added was methanol: acetonitrile 1:1(v/v), containing 1% formic acid.
The migration of single cells to the tip of the capillary glass tube is shown in FIG. 3, and the resulting mass spectrum is shown in FIG. 4. The lipid substances detected were mainly glycerophosphorylcholine, triglyceride, etc. The detection result shows that the method can simply and efficiently realize the detection and analysis of lipid substances in single cells.
Example 2 lipid analysis of MCF7 cells after Single photochemical derivatization
Fixation of cells: cultured cells were transferred to PBS. The cells were resuspended in Phosphate Buffered Saline (PBS) containing 2.5% glutaraldehyde using 1200rpm centrifugation, twenty minutes later, centrifuged, harvested and resuspended in water.
Photochemical derivatization of cells: to the cell suspension, a solution of 2-acetylpyridine was added to a final concentration of 100 mM. In a quartz reaction cell (as shown in FIG. 2), UV chemistry was carried out using a UV lamp of 254nm for 8 minutes.
Preparing a capillary glass tube: capillary glass tubes with a tip diameter of 5 μm were drawn using a P1000 pin drawing machine.
Mass spectrometry analysis:
(1) adding 0.5 mu L of cell suspension (1-5 cells) into the capillary glass tube from the tail end of the capillary glass tube, selecting the capillary glass tube containing 1 cell under a microscope, and transferring the single derived MCF7 cell to the front end of the capillary glass tube by using-1.2 kV voltage.
(2) And waiting for the liquid in the capillary glass tube to evaporate.
(3) A voltage of- +1.8kV was applied while dropping an auxiliary droplet from the tip. The auxiliary drop added was methanol: acetonitrile 1:1(v/v), containing 1% formic acid.
The resulting mass spectrum is shown in FIG. 5. The lipid substances detected are mainly glycerophosphocholine, fatty acid, diglyceride, triglyceride, cholesterol oleate, etc. The detection result shows that the method can simply and efficiently realize the detection and analysis of the lipid substances in the derived single cells.
Meanwhile, the inventors compared the effect of performing the light derivatization reaction using 2-acetylpyridine (2AP), 3-acetylpyridine (3AP), and 4-acetylpyridine (4 AP). The results are shown in FIG. 6. From the viewpoint of the reaction yield, 2-acetylpyridine is most effective.
EXAMPLE 3 identification of the carbon-carbon double bond isomeric Structure of a photochemically-derived MCF7 intracellular Glycerol Phosphocholine (PC)
Fixation of cells: cultured cells were transferred to PBS. The cells were resuspended in Phosphate Buffered Saline (PBS) containing 2.5% glutaraldehyde using 1200rpm centrifugation, twenty minutes later, centrifuged, harvested and resuspended in water.
Photochemical derivatization of cells: to the cell suspension, a solution of 2-acetylpyridine was added to a final concentration of 100 mM. In a quartz reaction cell (as shown in FIG. 2), UV chemistry was carried out using a UV lamp of 254nm for 8 minutes.
Preparing a capillary glass tube: capillary glass tubes with a tip diameter of 5 μm were drawn using a P1000 pin drawing machine.
Mass spectrometry analysis:
(1) adding 0.5 mu L of cell suspension (1-5 cells) into the capillary glass tube from the tail end of the capillary glass tube, selecting the capillary glass tube containing 1 cell under a microscope, and transferring the single derived MCF7 cell to the front end of the capillary glass tube by using-1.2 kV voltage.
(2) And waiting for the liquid in the capillary glass tube to evaporate.
(3) A voltage of- +1.8kV was applied while dropping an auxiliary droplet from the tip. The auxiliary drop added was methanol: acetonitrile 1:1(v/v), containing 1% formic acid. Meanwhile, the structure of specific lipid is analyzed by tandem mass spectrometry.
The mass spectrum obtained is shown in FIG. 7, using PC 34:1 as an example. There are three double bond isomers of PC 34: 1: PC 16: 0-18: 1 (. DELTA.8), PC 16: 0-18: 1 (. DELTA.9) and PC 16: 0-18: 1 (. DELTA.11). After photochemical derivatization, PC 16: 0-18: 1 (. DELTA.8) gives a pair of diagnostic ions with charge-to-mass ratios of 636 and 725, by tandem mass spectrometry. After photochemical derivatization, PC 16: 0-18: 1 (. DELTA.9) gave a pair of diagnostic ions with charge-to-mass ratios of 650 and 739 by tandem mass spectrometry. After photochemical derivatization, PC 16: 0-18: 1 (. DELTA.11) gives a pair of diagnostic ions with charge-to-mass ratios of 678 and 767 by tandem mass spectrometry. In the mass spectrogram, the relative content ratios of the three isomers in the cell can be obtained by calculating the strength ratios of the three pairs of diagnostic ions.
EXAMPLE 4 identification of the carbon-carbon double bond isomeric structures of a single photochemically-derivatized MCF7 intracellular Fatty Acid (FA), Diglyceride (DAG), Triglyceride (TAG), Cholesterol oleate (CE)
Fixation of cells: cultured cells were transferred to PBS. The cells were resuspended in Phosphate Buffered Saline (PBS) containing 2.5% glutaraldehyde using 1200rpm centrifugation, twenty minutes later, centrifuged, harvested and resuspended in water.
Photochemical derivatization of cells: to the cell suspension, a solution of 2-acetylpyridine was added to a final concentration of 100 mM. In a quartz reaction cell (as shown in FIG. 2), UV chemistry was carried out using a UV lamp of 254nm for 8 minutes.
Preparing a capillary glass tube: capillary glass tubes with a tip diameter of 5 μm were drawn using a P1000 pin drawing machine.
Mass spectrometry analysis:
(1) adding 0.5 mu L of cell suspension (1-5 cells) into the capillary glass tube from the tail end of the capillary glass tube, selecting the capillary glass tube containing 1 cell under a microscope, and transferring the single derived MCF7 cell to the front end of the capillary glass tube by using-1.2 kV voltage.
(2) And waiting for the liquid in the capillary glass tube to evaporate.
(3) A voltage of- +1.8kV was applied while dropping an auxiliary droplet from the tip. The drops added were methanol: acetonitrile: chloroform 45:45:10(v/v), contains 1% formic acid. Meanwhile, the structure of specific lipid is analyzed by tandem mass spectrometry.
For Fatty Acid (FA), FA 18:1 is used as an example, and the obtained mass spectrum is shown in FIG. 8 (a). There are two double bond isomers of FA 18:1, FA 18:1 (. DELTA.9) and FA 18:1 (. DELTA.11). FA 18:1 (. DELTA.9) after photochemical derivatization, was used to obtain a pair of diagnostic ions with a charge-to-mass ratio of 232,262 by tandem mass spectrometry. FA 18:1 (. DELTA.11) after photochemical derivatization, was used to obtain a pair of diagnostic ions with a charge-to-mass ratio of 204,290 by tandem mass spectrometry. In the mass spectrogram, the relative content ratio of the two isomers in the cell can be obtained by calculating the intensity ratio of the two pairs of diagnostic ions.
For Diglyceride (DAG), taking DAG 18: 1-18: 1 as an example, the mass spectrum obtained is shown in FIG. 8 (b). There are two types of double bond isomers of DAG 18: 1-18: 1, containing C18:1 (. DELTA.9) and C18:1 (. DELTA.11). After photochemical derivatization, a pair of diagnostic ions with the charge-to-mass ratio of 493,600 can be obtained by tandem mass spectrometry, wherein the pair of diagnostic ions contains C18:1 (delta 9). After photochemical derivatization, a pair of diagnostic ions with a charge-to-mass ratio of 521,628 can be obtained by tandem mass spectrometry, containing C18:1 (. DELTA.11). In the mass spectrogram, the relative content ratio of the two isomers in the cell can be obtained by calculating the intensity ratio of the two pairs of diagnostic ions.
For cholesterol oleate (CE), the mass spectrum obtained is shown in FIG. 8(c), using CE 18:1 as an example. There are two double bond isomers of CE 18:1, CE 18:1 (. DELTA.9) and CE 18:1 (. DELTA.11). After photochemical derivatization, CE 18:1(Δ 9) gave a diagnostic ion with a charge to mass ratio of 630 by tandem mass spectrometry. After photochemical derivatization of CE 18:1 (. DELTA.11), diagnostic ions were obtained by tandem mass spectrometry with a charge-to-mass ratio of 658. A cholesterol peak (charge to mass ratio 369) characteristic of CE can be observed in the mass spectrum. In the mass spectrogram, the relative content ratio of the two isomers in the cell can be obtained by calculating the strength ratio of the two diagnostic ions.
For Triglycerides (TAG), taking TAG 18: 1-18: 1 as an example, the resulting mass spectrum is shown in FIG. 8 (d). There are two double bond isomers of TAG 18: 1-18: 1, containing C18:1 (. DELTA.9) and C18:1 (. DELTA.11). After photochemical derivatization, a pair of diagnostic ions with the charge-to-mass ratio of 493,864 can be obtained by tandem mass spectrometry, wherein the pair of diagnostic ions contains C18:1 (delta 9). After photochemical derivatization, a pair of diagnostic ions with a charge-to-mass ratio of 521,892 can be obtained by tandem mass spectrometry, containing C18:1 (. DELTA.11). In the mass spectrogram, the relative content ratio of the two isomers in the cell can be obtained by calculating the intensity ratio of the two pairs of diagnostic ions.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (12)
1. A method of single-cell mass spectrometry comprising:
carrying out cell fixation and dilution treatment on cells to be detected so as to obtain a pretreated cell suspension;
injecting the pretreated cell suspension into a cavity from the tail end of the capillary, wherein an electrode is inserted into the cavity;
applying a voltage to the electrodes such that cells migrate to the capillary tip;
allowing the liquid in the capillary to evaporate completely;
and dropping auxiliary liquid into the tip of the capillary, enabling the auxiliary liquid to enter the capillary and contact with the cells to extract a target object, and simultaneously applying voltage to the electrode to perform electrospray so as to enable the auxiliary liquid containing the target object to be ionized, to be ejected from the tip, to enter a sample inlet of a mass spectrometer, and to perform mass spectrometry.
2. The method of claim 1, wherein the target is a lipid material having a carbon-carbon double bond, the method further comprising the steps of:
and carrying out photochemical derivatization treatment on the cell suspension subjected to the cell fixing treatment, and then carrying out the dilution treatment on the obtained cell suspension.
3. The method of claim 2, wherein the derivatizing agent used in the photochemical derivatizing treatment is selected from the group consisting of water-soluble carbonyl compounds.
4. The method of claim 2, wherein the photochemical derivatization is performed using a derivatization reagent selected from the group consisting of 2-acetylpyridine, 3-acetylpyridine, and 4-acetylpyridine.
5. The method of claim 3, wherein the photochemical derivatization process comprises:
and mixing the cell suspension with the derivatization reagent, and irradiating for 1-15 minutes under 250-260 nm ultraviolet light.
6. The method of claim 1, wherein the cell fixative used in the cell fixation treatment is selected from the group consisting of aldehydic chemicals.
7. The method of claim 1, wherein the cell fixation solution used in the cell fixation treatment is selected from paraformaldehyde or glutaraldehyde.
8. The method of claim 1, wherein the number of cells in said pretreated cell suspension is 2 x 103～1×104And each/mL, wherein the volume of the pretreated cell suspension added into the capillary tube is 0.3-0.6 mu L.
9. The method of claim 1, wherein the capillary tip has an outer diameter that is smaller than the diameter of the cells in the pretreated cell suspension.
10. The method of claim 1, wherein the capillary tip has an outer diameter of 3 to 10 μm.
11. The method of claim 1, wherein the auxiliary liquid is selected from at least one of methanol, ethanol, acetonitrile, acetone, chloroform, isopropanol, and water.
12. The method of claim 1, wherein the target is selected from at least one of choline glycerophosphate, a diglyceride, a triglyceride, a fatty acid, and a cholesterol oleate.
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| CN111965093B (en) * | 2020-10-26 | 2021-02-05 | 宁波华仪宁创智能科技有限公司 | Single cell mass spectrometry device and method |
| CN112444583B (en) * | 2021-02-01 | 2021-05-07 | 宁波大学 | Cell detection device and method based on paper-based discharge technology |
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