CN112748199A - Cell lipidomics analysis method based on liquid chromatography tandem mass spectrometry - Google Patents
Cell lipidomics analysis method based on liquid chromatography tandem mass spectrometry Download PDFInfo
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- 238000004458 analytical method Methods 0.000 title claims abstract description 27
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 16
- 239000011259 mixed solution Substances 0.000 claims abstract description 26
- 239000000243 solution Substances 0.000 claims abstract description 22
- 150000002632 lipids Chemical class 0.000 claims abstract description 15
- 239000000126 substance Substances 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 7
- 239000012074 organic phase Substances 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- 239000006285 cell suspension Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 239000011261 inert gas Substances 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 abstract description 3
- -1 lipid compounds Chemical class 0.000 abstract description 3
- 238000007405 data analysis Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 29
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- 239000007789 gas Substances 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- BDXAHSJUDUZLDU-UHFFFAOYSA-N methyl nonadecanoate Chemical group CCCCCCCCCCCCCCCCCCC(=O)OC BDXAHSJUDUZLDU-UHFFFAOYSA-N 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 230000037237 body shape Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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Abstract
The invention discloses a cell lipidomics analysis method based on liquid chromatography tandem mass spectrometry, which comprises the steps of crushing a cell solution, and adding a standard substance of an isotope internal standard to obtain a first mixed solution; adding the two extracting solutions into the first mixed solution, mixing in a vortex mode, and standing; standing, centrifuging to obtain an upper organic phase, drying, and mixing and redissolving by using a redissolving solution to obtain a sample; then carrying out lipidomics data acquisition on the sample by using an ultra-performance liquid chromatography-mass spectrometer; performing data analysis by using software to obtain an analysis result; the invention can detect the lipid component of the labeled cells and the mass isotope isomer thereof; it is also possible to compare different types of cellular properties according to the type and amount of lipid compounds in the cells and to achieve an exact match of the isotopologues of the same lipid source.
Description
Technical Field
The invention relates to a cell lipidomics analysis method based on liquid chromatography-tandem mass spectrometry, and belongs to the technical field of cell lipidomics analysis methods.
Background
The cell is the basic unit of organism structure and function, the body shape is tiny, the shape is various; the cell is mainly composed of nucleus and cytoplasm, and has cell membrane on the surface and cell wall outside the cell membrane of higher plant. The chemical substances composing the cells are divided into inorganic substances and organic substances, the organic substances in the cells are more than thousands of kinds, and account for more than 90% of the dry weight of the cells, and the organic substances mainly comprise four major molecular compositions, namely proteins, nucleic acids, lipids and saccharides.
The existing detection methods only aim at individual cells, but the existing situation of lipid in more cells is not known, and the detection of the lipid component and the mass isotope isomer of the lipid component in the cells by detecting a label cannot be realized.
Disclosure of Invention
Aiming at the existing technical problems, the invention aims to: provides a cell lipidomics analysis method based on liquid chromatogram tandem mass spectrum, which can detect and mark cell lipid components and mass isotope isomers thereof.
The technical solution of the invention is realized as follows: a cell lipidomics analysis method based on liquid chromatography tandem mass spectrometry comprises the following steps:
the method comprises the following steps: extracting cells to be detected, and adjusting the concentration of the cells to be detected to obtain a preparation solution;
step two: carrying out ultrasonic disruption on the preparation solution to obtain a cell suspension;
step three: adding a standard substance of an isotope internal standard into the cell suspension, and performing vortex mixing to obtain a first mixed solution;
step four: adding a first extracting solution into the first mixed solution, and carrying out vortex mixing to obtain a second mixed solution;
step five: adding a second extracting solution into the second mixed solution, and carrying out vortex mixing to obtain a third mixed solution;
step six: standing the third mixed solution at room temperature, centrifuging to obtain an upper organic phase, drying, and mixing with a redissolving solution for redissolving to obtain a sample;
step seven: performing lipidomics data acquisition on the sample with an analytical instrument; attributing isotope isomers belonging to the same labeled lipid molecule, and performing mass isotope isomer distribution analysis on the isotope isomers;
step eight: and analyzing the data acquired in the step seven by using software to obtain an analysis result.
In one embodiment of the invention, the redissolution solution in the sixth step is methanol, isopropanol or acetonitrile.
In one embodiment of the present invention, the drying method in the sixth step is a constant temperature evaporation method or an inert gas blowing method.
In one embodiment of the invention, the analysis instrument in the seventh step is an ultra high performance liquid chromatography-mass spectrometry instrument or a chromatography combination type quadrupole-orbitrap mass spectrometry.
In one embodiment of the present invention, the first extract in the fourth step is methanol.
In one embodiment of the present invention, the second extract in the fifth step is methyl tert-butyl ether or dichloromethane.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
the cell lipidomics analysis method based on the liquid chromatography-tandem mass spectrometry can detect the labeled cell lipid component and the mass isotope isomer thereof; it is also possible to compare different types of cellular properties according to the type and amount of lipid compounds in the cells and to achieve an exact match of the isotopologues of the same lipid source.
Drawings
The technical scheme of the invention is further explained by combining the accompanying drawings as follows:
FIG. 1 is a step diagram of a method for analyzing cell lipidomics based on liquid chromatography-tandem mass spectrometry.
Detailed Description
The invention is described below with reference to the accompanying drawings.
Referring to fig. 1, the first embodiment:
a cell lipidomics analysis method based on liquid chromatography tandem mass spectrometry comprises the following steps:
the method comprises the following steps: extracting cells to be detected, and adjusting the concentration of the cells to be detected to obtain a preparation solution;
step two: carrying out ultrasonic disruption on the preparation solution to obtain a cell suspension;
step three: adding a standard substance of an isotope internal standard into the cell suspension, and performing vortex mixing to obtain a first mixed solution; isotope internal standards are one or more of CA-d5, DCA-d5, LCA-d4, GLCA-d4, TCA-d4, TLCA-d5, TCDCA-d5 and TUDCA-d 5;
step four: adding methanol into the first mixed solution, and carrying out vortex mixing to obtain a second mixed solution;
step five: adding methyl tert-butyl ether into the second mixed solution, and performing vortex mixing to obtain a third mixed solution;
step six: standing the third mixed solution at room temperature, centrifuging to obtain an upper organic phase, drying by using a constant-temperature steaming method, and mixing and redissolving by using methanol to obtain a sample;
step seven: carrying out lipidomics data acquisition on the sample by using an ultra-high performance liquid chromatography-mass spectrometer; attributing isotope isomers belonging to the same labeled lipid molecule, and performing mass isotope isomer distribution analysis on the isotope isomers;
wherein the chromatographic conditions are as follows:
a chromatographic column: synergi Polar-RP (2.0X 50mm,5 μm); column temperature: 50 ℃; flow rate: 800 muL/min;
mobile phase A: 0.1% aqueous formic acid; mobile phase B: 0.1% formic acid in methanol.
Wherein the mass spectrum conditions are as follows: the ion source parameters are Curtain Gas (CUR)20, Collision Gas (CAD)9, ion spray voltage-4500V, source temperature 600 ℃, CUR 25.0, GS 160.0, and GS 265.0.
Step eight: and analyzing the data acquired in the step seven by using software to obtain an analysis result.
Please refer to fig. 1, embodiment two:
a cell lipidomics analysis method based on liquid chromatography tandem mass spectrometry comprises the following steps:
the method comprises the following steps: extracting cells to be detected, and adjusting the concentration of the cells to be detected to obtain a preparation solution;
step two: carrying out ultrasonic disruption on the preparation solution to obtain a cell suspension;
step three: adding a standard substance of an isotope internal standard into the cell suspension, and performing vortex mixing to obtain a first mixed solution; the isotope internal standard is n-nonadecanoic acid methyl ester; isotope internal standards are one or more of CA-d5, DCA-d5, LCA-d4, GLCA-d4, TCA-d4, TLCA-d5, TCDCA-d5 and TUDCA-d 5;
step four: adding methanol into the first mixed solution, and carrying out vortex mixing to obtain a second mixed solution; the concentration of the n-nonadecanoic acid methyl ester is 1.5 mg/mL;
step five: adding dichloromethane into the second mixed solution, and performing vortex mixing to obtain a third mixed solution;
step six: standing the third mixed solution at room temperature, centrifuging to obtain an upper organic phase, drying by using helium, and mixing and redissolving by using isopropanol to obtain a sample;
step seven: carrying out lipidomics data acquisition on the sample by using a chromatographic combined type quadrupole-orbital trap mass spectrum; attributing isotope isomers belonging to the same labeled lipid molecule, and performing mass isotope isomer distribution analysis on the isotope isomers;
wherein the chromatographic conditions are as follows:
chromatographic column Waters UPLC HSS T3(2.1mm 50mm,1.8 μm) + on-line filter; column temperature: 55 ℃; sample introduction amount: 10 mu L of the solution; the flow rate is 0.8 mL/min;
mobile phase A: formic acid, ammonium acetate and pure water; mobile phase B: formic acid + ammonium acetate + methanol;
wherein the mass spectrum conditions are as follows: ESI source, positive ion mode, heating temperature: 300 ℃, sheath gas flow rate): 45arb, assist gas flow rate: 20arb, purge gas flow rate: 1arb, spray voltage: 3.5KV, temperature of ion transfer tube: 400 ℃; the first-level scanning range is 200-2000;
step eight: and analyzing the data acquired in the step seven by using software to obtain an analysis result.
The cell lipidomics analysis method based on the liquid chromatography-tandem mass spectrometry can detect the labeled cell lipid component and the mass isotope isomer thereof; it is also possible to compare different types of cellular properties according to the type and amount of lipid compounds in the cells and to achieve an exact match of the isotopologues of the same lipid source.
The embodiments are only for illustrating the technical concept and characteristics of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be covered in the scope of the present invention.
Claims (6)
1. A cell lipidomics analysis method based on liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps:
the method comprises the following steps: extracting cells to be detected, and adjusting the concentration of the cells to be detected to obtain a preparation solution;
step two: carrying out ultrasonic disruption on the preparation solution to obtain a cell suspension;
step three: adding a standard substance of an isotope internal standard into the cell suspension, and performing vortex mixing to obtain a first mixed solution;
step four: adding a first extracting solution into the first mixed solution, and carrying out vortex mixing to obtain a second mixed solution;
step five: adding a second extracting solution into the second mixed solution, and carrying out vortex mixing to obtain a third mixed solution;
step six: standing the third mixed solution at room temperature, centrifuging to obtain an upper organic phase, drying, and mixing with a redissolving solution for redissolving to obtain a sample;
step seven: performing lipidomics data acquisition on the sample with an analytical instrument; attributing isotope isomers belonging to the same labeled lipid molecule, and performing mass isotope isomer distribution analysis on the isotope isomers;
step eight: and analyzing the data acquired in the step seven by using software to obtain an analysis result.
2. The method of claim 1 for the cytolipidomics analysis based on liquid chromatography-tandem mass spectrometry, wherein: and in the sixth step, the redissolution is methanol, isopropanol or acetonitrile.
3. The method of claim 1 for the cytolipidomics analysis based on liquid chromatography-tandem mass spectrometry, wherein: and the drying method in the sixth step is a constant-temperature evaporation method or an inert gas blowing method.
4. The method of claim 1 for the cytolipidomics analysis based on liquid chromatography-tandem mass spectrometry, wherein: and the analyzer in the seventh step is an ultra-high performance liquid chromatography-mass spectrometer or a chromatography combined type quadrupole-orbitrap mass spectrometer.
5. The method of claim 1 for the cytolipidomics analysis based on liquid chromatography-tandem mass spectrometry, wherein: the first extracting solution in the fourth step is methanol.
6. The method of claim 1 for the cytolipidomics analysis based on liquid chromatography-tandem mass spectrometry, wherein: and in the fifth step, the second extracting solution is methyl tert-butyl ether or dichloromethane.
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