CN114609225B - Small-cell lipidomic analysis method - Google Patents
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Abstract
The invention discloses a high-sensitivity high-flux lipidomic analysis method adopting a microprobe sampling-multichannel chip nano-spraying-high-resolution mass spectrometry technology aiming at a small number of cells (15-25). According to the method, 15-25 animal cells are accurately sampled under a microscope by adopting a capillary microprobe, lipid extraction is directly carried out in an orifice plate, the animal cells directly enter a high-resolution mass spectrum for analysis through a multichannel chip nano-spray ion source, first-stage mass spectrum information is acquired by adopting a spliced sectional scanning mode, and qualitative lipid is obtained after database matching. The invention realizes that the single analysis only needs 2 minutes of flux, can identify more than 500 lipids from 20 breast cancer cells, has the characteristics of simple flow, high sensitivity and high analysis flux, and is suitable for high-sensitivity and high-flux analysis of rare cells which are difficult to obtain, such as stem cells and circulating tumor cell lipidosomes.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a high-sensitivity high-flux lipidomic analysis method adopting a microprobe sampling-multichannel chip nano-spraying-high-resolution mass spectrometry technology for a small number of cells.
Background
Metabonomics is the systematic study of the response of a living organism to multiple dynamic metabolites in the body when subjected to intrinsic genetic mutations, pathophysiological changes, or external environmental stimuli. The cell is a basic unit of the structure and the function of a living body, and the metabonomics research taking the cell as a target is helpful for revealing the process and the rule of the life activity. Traditional metabonomics studies to increase the number of quantifiable metabolites in cell extracts, metabonomic analysis is typically performed using a large number of cells (e.g., millions of cells). However, reducing the number of cells required for metabonomics would be beneficial for many studies, such as the rarity and difficulty in obtaining stem cells, circulating tumor cells, and primary cells obtained from tissue; on the other hand, if the metabonomics research of a small number of cells is feasible, a large number of cells do not need to be cultured, so that the manpower and material resources can be saved, the test period can be shortened, the test cost can be greatly reduced, and meanwhile, the cell biology research is more convenient. Therefore, it is of great importance to develop a metabonomic analysis method for a small number of cells.
The challenge of small numbers of cell metabonomics analysis is the high sensitivity and high throughput detection of metabolites. The liquid chromatography-mass spectrometry (LC-MS) technology has become the mainstream technology of the current metabonomics analysis research due to the characteristics of high sensitivity, strong specificity, diversity and the like. However, LC chromatographic separation prior to mass spectrometry is often time consuming and high throughput analysis is difficult to achieve. In addition, conventional electrospray ionization sources (ESI) generally have strong matrix interference and ion suppression effects, and cannot meet the sensitivity requirements of small amounts of cellular metabolite analysis. On the other hand, the acquisition of a small amount of cells in the reported research of a small amount of cell metabonomics still needs to be obtained from dilution and counting of constant cells, which is very unfavorable for the research of rare cells which are difficult to culture on a large scale, and complicated and time-consuming sample pretreatment steps such as cell lysis, metabolite extraction and the like can not only cause the loss of samples, but also limit the improvement of analysis flux.
Aiming at the difficult problem existing in the current research of a small amount of cell metabonomics, the invention adopts the accurate sampling of 15-25 cells by using the microprobe, and the spray solvent is directly added into the pore plate for extracting the lipid, so that the complex sample pretreatment is not needed, and the sample loss is avoided; the nano-liter electrospray ionization source for direct sample injection has high analysis flux while reducing matrix effect and improving detection sensitivity; the adopted high-resolution mass spectrum spliced type segmented scanning mode can not only remarkably improve the detection sensitivity, but also maintain higher quality precision, and greatly improve the accuracy of metabolite qualitative.
Disclosure of Invention
The invention establishes a high-sensitivity high-flux analysis method based on a microprobe sampling-multichannel chip nano-spraying-high-resolution mass spectrometry technology for realizing the metabonomics analysis of a small number of cells. The method has the advantages of accurate cell sampling, no need of sample pretreatment, high sensitivity, high throughput and the like.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
(1) Drawing a capillary microprobe by using a needle drawing instrument (P-1000,Sutter Instrument,Novato,U.S.A.) and fixing the capillary microprobe on a movable slide rail connected with a three-dimensional micro-movable operation platform, precisely moving the capillary microprobe to the upper part of a target cell by using a microscope, sucking 15-25 animal cells under the action of negative pressure, and taking 0.4-0.6min in the sampling process;
(2) Injecting 15-25 drawn animal cells into 96-384 well plate via injection pump, adding 10-20 μl spray solvent for lipid extraction, and mixing with chloroform under positive ion mode: methanol: isopropanol (1:2:4, v/v/v), 5mM ammonium formate and 0.2% formic acid as modifiers; the composition of the negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v), 5mM ammonium acetate as modifier; both contain 12 lipid internal standards, including LPC 12:0、PC(15:0/15:0)、PE(15:0/15:0)、PG(15:0/15:0)、PA(17:0/17:0)、PS(16:0/16:0-d62)、Cer(d18:1/17:0)、SM(d18:1/12:0)、TG(15:0/15:0/15:0)、DG(12:0/12:0)、ChoE 17:0、FFA 18:0-d3, internal standard concentrations of 0.01-0.05 μg/ml. Standing the mixed solution at 0-4deg.C for 0.5-1min to complete lipid extraction;
(3) The lipid extraction sample directly enters a high-resolution mass spectrum for analysis through an automatic sampler of a multichannel chip nano-spray ion source, and the first-stage mass spectrum information is acquired by adopting a spliced sectional scanning mode. The operating conditions were as follows:
① Multichannel chip nano-spray ion source conditions: d chip (nozzle inner diameter 4.1 μm); the spraying voltage in the positive ion mode is +1.5kV, and the spraying voltage in the negative ion mode is-1.8 kV; the air pressure was 0.6psi; the temperature of the sample injection chamber is 4 ℃; the sample injection volume is 5 mu L;
② High resolution combined quaternary rod-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; setting a first-stage m/z spliced segmented scanning window in a positive ion mode as 290-390、380-480、470-570、560-610、600-650、640-690、680-730、720-770、760-810、800-850、840-940、930-1030、1020-1120、1110-1210、1200-1300Da, and setting 150-250、240-340、330-430、420-520、510-610、600-650、640-690、680-730、720-770、760-810、800-850、840-940、930-1030、1020-1120、1110-1210、1200-1300Da; micro-scanning windows in a negative ion mode as 3; the resolution is 240K; the injection time was set to 200ms; the dynamic gain is controlled to be 1e 6, and the acquisition time is 0.6min;
(4) Repeating the step (1-3) for 6-10 times; and (3) deriving primary mass spectrum data by adopting Xcalibur software, and carrying out peak matching on the data to obtain a characteristic ion total peak table containing m/z and peak intensity. Subtracting the characteristic ions with solvent blank, signal-to-noise ratio less than 10 and occurrence frequency less than 80% in 6-10 repetitions to obtain a stable characteristic ion list, matching the characteristic ion list with the accurate m/z in LIPID MAPS databases, and setting the quality precision to be +/-3 ppm to obtain a qualitative lipid list;
(5) To a macrocell (1X 10 6-5×106) dish was added 1-2mL of lipid extraction solvent whose composition in positive ion mode was chloroform: methanol: isopropanol (1:2:4, v/v/v), with 5mM ammonium formate and 0.2% formic acid as modifier; the negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v), with 5mM ammonium acetate as modifier. The two extraction solvents contain 12 lipid internal standards, including LPC 12:0、PC(15:0/15:0)、PE(15:0/15:0)、PG(15:0/15:0)、PA(17:0/17:0)、PS(16:0/16:0-d62)、Cer(d18:1/17:0)、SM(d18:1/12:0)、TG(15:0/15:0/15:0)、DG(12:0/12:0)、ChoE 17:0、FFA 18:0-d3, internal standard concentration of 0.01-0.05 μg/ml. The supernatant of the mixed solution after oscillation, standing and centrifugation is the cell lipid extract, and the cell lipid extract directly enters a high-resolution mass spectrum for analysis through an automatic sampler of a multichannel chip nano-spray ion source, and the first-stage mass spectrum information is acquired by adopting a spliced sectional scanning mode. The samples were subjected to 6-10 replicates under the same conditions as (3).
(6) And (3) deriving the primary mass spectrum data of the step (5) by using Xcalibur software, and performing peak matching on the data to obtain a characteristic ion total peak table containing m/z and peak intensity. After subtracting the characteristic ions with solvent blank, signal-to-noise ratio less than 10 and occurrence frequency less than 80% in 6-10 repetitions, obtaining a stable characteristic ion list, matching the characteristic ion list with the accurate m/z in LIPID MAPS databases, setting the mass accuracy to be +/-3 ppm, and taking the matched m/z as a lipid list for PRM secondary mass spectrum acquisition. The operating conditions were as follows:
① The conditions of the multichannel chip nano-spray ion source are the same as those of the (3);
② High resolution combined quaternary rod-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; resolution is 120K; the injection time was 200ms; the dynamic gain is controlled to be 5e 5, and the isolation window is 0.4m/z; the collision energy is 10eV, 20eV, 25eV, 30eV and 40eV;
The characteristic ions, neutral losses and/or fatty acyl ion fragments in the secondary mass spectrogram are checked one by one, and the lipids are subjected to detailed structural annotation, so that a constant cell lipid database comprising MS and/or MS/MS information is obtained. Lipids with fragmentation information were characterized using MS, MS/MS; otherwise, only MS is used for characterization.
(7) Matching m/z of a small amount of cell qualitative lipid with accurate m/z in a constant cell lipid database, setting the mass precision to be +/-3 ppm, and if the mass precision can be matched with lipid containing MS and MS/MS information in the constant cell lipid database, setting the qualitative grade to be Level 1; if the lipid can be matched with the lipid containing MS information in the constant cell lipid database, the qualitative grade is Level 2; if it cannot match the lipids in the macrocell lipid database, the qualitative rating is Level 3. If the lipid with the qualitative grade of Level 3 accounts for less than 10% of all qualitative lipids, the qualitative method which only depends on a small amount of cells to be matched with the accurate m/z in LIPID MAPS database in the first-order m/z is considered to be reliable, and the follow-up study does not need to depend on constant cells to assist in qualitative analysis, so that the method can be directly used for metabonomics analysis of rare cells.
The invention realizes that the single analysis only needs 2 minutes of flux, can identify more than 500 lipids from 20 breast cancer cells, has the characteristics of simple flow, high sensitivity and high analysis flux, and is suitable for high-sensitivity and high-flux analysis of rare cells which are difficult to obtain, such as stem cells and circulating tumor cell lipidosomes.
The invention adopts the microprobe to accurately sample 15-25 cells, and spray solvent is directly added into the pore plate to finish lipid extraction, so that sample pretreatment is not needed, and sample loss is avoided as far as possible; the nano-liter electrospray ionization source for direct sample injection has high analysis flux while reducing matrix effect and improving detection sensitivity; the adopted high-resolution mass spectrum spliced type segmented scanning mode can further improve the detection sensitivity, can also keep higher quality precision, and greatly improves the accuracy of metabolite qualitative.
Drawings
FIG. 1 (A) lipid characterization strategy for a small number of cells; (B) Schematic of the construction flow of the constant cell lipid database.
FIG. 2 is a schematic illustration of an m/z quality window setup for a tiled segmented scan.
Figure 3 positive and negative ion patterns small numbers (20) of cells characterize different classes of lipid percentages.
FIG. 4 is a schematic representation of the results of qualitative lipid validation of 20 MCF7 cells in a macrocell lipid database.
FIG. 5 principal component analysis score of lipids with p <0.05 in MCF7, MHCC97H and VCaP three cancer cells.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the attached drawing figures: the embodiments and specific operation procedures are given on the premise of the technical scheme of the invention, but the protection scope of the invention is not limited to the following embodiments.
Example 1
A flow chart established by a small amount of cell lipidomics high-sensitivity high-throughput analysis method based on a microprobe sampling-multichannel chip nano-jet-high-resolution mass spectrometry technology is shown in fig. 1, and the specific implementation steps are as follows:
(1) 20 MCF7 cell samples:
Drawing a capillary micro-probe (the inner diameter of a tip is 5-10 mu m) by using a needle drawing instrument (P-1000,Sutter Instrument,Novato,U.S.A.) and fixing the capillary micro-probe on a movable slide rail connected with a three-dimensional micro-movable operation platform, precisely moving the capillary micro-probe to the upper part of a target cell by using a microscope, sucking 20 breast cancer cells (MCF 7) under the action of negative pressure, and repeatedly sampling for 6 times;
(2) Lipid extraction:
the 20 MCF7 cells were injected into a 96-well plate by a syringe pump, and 15. Mu.L of spray solvent was added for lipid extraction, and the composition in positive ion mode was chloroform: methanol: isopropanol (1:2:4, v/v/v), containing 5mM ammonium formate and 0.2% formic acid as modifier in final volume concentration; the composition of the negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v), containing 5mM ammonium acetate as modifier; both spray solvents contained 12 lipid internal standards, including LPC 12:0、PC(15:0/15:0)、PE(15:0/15:0)、PG(15:0/15:0)、PA(17:0/17:0)、PS(16:0/16:0-d62)、Cer(d18:1/17:0)、SM(d18:1/12:0)、TG(15:0/15:0/15:0)、DG(12:0/12:0)、ChoE 17:0、FFA 18:0-d3, internal standard concentrations of 0.01-0.05 μg/ml. Standing the mixed solution at 4deg.C for 1min to complete lipid extraction;
(3) Direct sample injection-high resolution mass spectrometry acquisition:
the lipid extract sample directly enters a high-resolution mass spectrum for analysis through an automatic sampler of a multichannel chip nano-spray ion source, and the first-stage mass spectrum information is acquired by adopting a spliced type sectional scanning mode. The m/z mass window segmented scan setup is shown in fig. 2.
① Multichannel chip nano-spray ion source conditions: d chip (nozzle inner diameter 4.1 μm); the spraying voltage in the positive ion mode is +1.5kV, and the spraying voltage in the negative ion mode is-1.8 kV; the air pressure was 0.6psi; the temperature of the sample injection chamber is 4 ℃; the sample injection volume is 5 mu L;
② High resolution combined quaternary rod-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; setting a quality window of the spliced segmented scanning in a positive ion mode as 290-390、380-480、470-570、560-610、600-650、640-690、680-730、720-770、760-810、800-850、840-940、930-1030、1020-1120、1110-1210、1200-1300Da, and setting a quality window of the spliced segmented scanning in a negative ion mode as 150-250、240-340、330-430、420-520、510-610、600-650、640-690、680-730、720-770、760-810、800-850、840-940、930-1030、1020-1120、1110-1210、1200-1300Da; micro-scanning as 3; the resolution is 240K; the injection time was set to 200ms; the dynamic gain is controlled to be 1e 6, and the acquisition time is 0.6min;
(4) Lipid characterization:
And (3) deriving primary mass spectrum data by adopting Xcalibur software, and carrying out peak matching on the data to obtain a characteristic ion total peak table containing m/z and peak intensity. After subtracting the characteristic ions with solvent blank, signal-to-noise ratio less than 10 and frequency of occurrence lower than 80% in 6 repetitions, obtaining a stable characteristic ion list, matching the characteristic ion list with the accurate m/z in LIPID MAPS database, setting the quality precision to be +/-3 ppm, and obtaining qualitative lipid, see tables 1, 2 and figure 3;
(5) Constant (5×10 6) MCF7 cell lipid extraction:
5×10 6 MCF7 cell samples were placed in a petri dish, and after three washing steps with PBS, 1ml of lipid extraction solvent was added, and the composition in positive ion mode was chloroform: methanol: isopropanol (1:2:4, v/v/v) containing 5mM ammonium formate and 0.2% formic acid as modifier; the composition of the negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v), containing 5mM ammonium acetate as modifier; the two extraction solvents both contain 12 lipid internal standards, including LPC 12:0、PC(15:0/15:0)、PE(15:0/15:0)、PG(15:0/15:0)、PA(17:0/17:0)、PS(16:0/16:0-d62)、Cer(d18:1/17:0)、SM(d18:1/12:0)、TG(15:0/15:0/15:0)、DG(12:0/12:0)、ChoE 17:0、FFA 18:0-d3, internal standard concentrations of 0.01-0.05 μg/ml. Transferring the mixed solution into a 2ml EP tube, swirling for 10s, shaking for 10min, standing for 10min at 4 ℃, centrifuging for 10min under 14000g condition, and taking 800 mu L of supernatant (namely lipid extract of macrocell) for sample injection analysis;
(6) Constant MCF7 cell direct injection-primary acquisition of high resolution mass spectrum:
Directly entering a constant cell lipid extract sample into a high-resolution mass spectrum for analysis through an automatic sampler of a multichannel chip nano-spray ion source, acquiring primary mass spectrum information by adopting a spliced type segmented scanning mode, and carrying out repeated analysis on the sample for 6 times, wherein the operation conditions are the same as (3);
(7) Constant MCF7 cell direct injection-high resolution mass spectrometry secondary acquisition:
the primary mass spectrum data of the step (6) are exported by Xcalibur software, and are subjected to peak matching by adopting an autonomous programming Python program, so that a characteristic ion total peak table containing m/z and peak intensity is obtained. After subtracting the characteristic ions with solvent blank, signal-to-noise ratio less than 10 and occurrence frequency less than 80% in 6 repetitions, obtaining a stable characteristic ion list, matching the characteristic ion list with accurate m/z in LIPID MAPS databases, setting mass accuracy to be +/-3 ppm, and taking the matched characteristic ions as a lipid list to carry out secondary mass spectrum acquisition of PRM. The operating conditions of the multi-channel chip nano-spray ion source-high resolution mass spectrum are as follows:
① The conditions of the multichannel chip nano-spray ion source are the same as those of the (3);
② High resolution combined quaternary rod-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; resolution is 120K; the injection time was 200ms; the dynamic gain is controlled to be 5e 5, and the isolation window is 0.4m/z; the collision energy is 10eV, 20eV, 25eV, 30eV and 40eV;
(8) Establishment of constant MCF7 cell lipid database:
The characteristic ions, neutral losses and/or fatty acyl ion fragments in the secondary mass spectrogram are checked one by one, and the lipids are subjected to detailed structural annotation, so that a constant cell lipid database comprising MS and/or MS/MS information is obtained. Lipids with fragmentation information were characterized using MS, MS/MS; otherwise, only MS is used for characterization.
(9) The 20 MCF7 cell qualitative lipids were validated based on a constant cell lipid database:
The m/z matching constant of the qualitative lipid of the 20 MCF7 cells MCF7 cell lipid database is set to be the accurate m/z, the quality precision is set to be +/-3 ppm, and if the quantitative lipid database can be matched with the lipid containing MS and MS/MS information in the constant cell lipid database, the qualitative grade is Level 1; if the lipid can be matched with the lipid containing MS information in the constant cell lipid database, the qualitative grade is Level 2; if the lipid is not matched with the lipid in the constant cell lipid database, the qualitative grade is Level 3, and the verification result is shown in fig. 4.
Example two
Different cancer cells (breast cancer cell MCF7, liver cancer cell mcc 97H, prostate cancer cell VCaP) were typed:
(1) Samples of 20 different cancer cells:
Drawing a capillary micro-probe (the inner diameter of a tip is 5-10 mu m) by using a needle drawing instrument (P-1000,Sutter Instrument,Novato,U.S.A.) and fixing the capillary micro-probe on a movable slide rail connected with a three-dimensional micro-movable operation platform, precisely moving the capillary micro-probe to the upper part of a target cell by means of a microscope, respectively sucking 20 breast cancer cells MCF7, liver cancer cells MHCC97H and prostate cancer cells VCaP under the action of negative pressure, and respectively sampling each cancer cell for 10 times;
(2) Lipid extraction:
20 MCF7 cells and MHCC97H, VCaP cells which are respectively sucked are injected into a 96-well plate through a syringe pump, 15 mu L of spray solvent is added for lipid extraction, and the composition of the lipid extraction is chloroform in a positive ion mode: methanol: isopropanol (1:2:4, v/v/v) containing 5mM ammonium formate and 0.2% formic acid as modifier; the composition of the negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v), containing 5mM ammonium acetate as modifier; both spray solvents contained 12 lipid internal standards, including LPC 12:0、PC(15:0/15:0)、PE(15:0/15:0)、PG(15:0/15:0)、PA(17:0/17:0)、PS(16:0/16:0-d62)、Cer(d18:1/17:0)、SM(d18:1/12:0)、TG(15:0/15:0/15:0)、DG(12:0/12:0)、ChoE 17:0、FFA18:0-d3, internal standard concentrations of 0.01-0.05 μg/ml. Standing the mixed solution at 4deg.C for 1min to complete lipid extraction;
(3) Direct sample injection-high resolution mass spectrometry acquisition:
the lipid extract sample directly enters a high-resolution mass spectrum for analysis through an automatic sampler of a multichannel chip nano-spray ion source, and the first-stage mass spectrum information is acquired by adopting a spliced type sectional scanning mode.
① Multichannel chip nano-spray ion source conditions: d chip (nozzle inner diameter 4.1 μm); the spraying voltage in the positive ion mode is +1.5kV, and the spraying voltage in the negative ion mode is-1.8 kV; the air pressure was 0.6psi; the temperature of the sample injection chamber is 4 ℃; the sample injection volume is 5 mu L;
② High resolution combined quaternary rod-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; the quality window of the spliced segmented scanning in the positive ion mode is set to 290-390、380-480、470-570、560-610、600-650、640-690、680-730、720-770、760-810、800-850、840-940、930-1030、1020-1120、1110-1210、1200-1300Da; microscan to be 3; the resolution is 240K; the injection time was set to 200ms; the dynamic gain is controlled to be 1e 6, and the acquisition time is 0.6min;
(4) Lipid characterization:
And (3) deriving primary mass spectrum data by adopting Xcalibur software, and carrying out peak matching on the data to obtain a characteristic ion total peak table containing m/z and peak intensity. Subtracting characteristic ions with solvent blank, signal-to-noise ratio less than 10 and occurrence frequency less than 80% in 10 repetitions to obtain a stable characteristic ion list, matching the characteristic ion list with accurate m/z in LIPID MAPS database, setting the quality accuracy to be +/-3 ppm, and respectively obtaining qualitatively-available lipids in different cancer cells (tables 1 and 2);
(5) Statistical analysis:
The qualitative lipid in different cancer cells is corrected to the total response intensity of the sample, the significant analysis is carried out by using a Student's t test, and the lipid with p <0.05 is selected for principal component analysis (figure 5), so that the different cancer cells are effectively distinguished, and the obvious difference of the lipid composition and the relative content of the different cancer cells is shown.
Table 1 positive ion mode qualitative lipid information in 20 MCF7 cells.
Table 2 negative ion mode qualitative lipid information in 20 MCF7 cells.
TABLE 1 qualitative lipid information in positive ion mode 20 MCF7 cells
TABLE 1 qualitative lipid information in positive ion mode 20 MCF7 cells (follow-up)
TABLE 1 qualitative lipid information in positive ion mode 20 MCF7 cells (follow-up)
TABLE 1 qualitative lipid information in positive ion mode 20 MCF7 cells (follow-up)
TABLE 1 qualitative lipid information in positive ion mode 20 MCF7 cells (follow-up)
TABLE 1 qualitative lipid information in positive ion mode 20 MCF7 cells (follow-up)
TABLE 1 qualitative lipid information in positive ion mode 20 MCF7 cells (follow-up)
TABLE 1 qualitative lipid information in positive ion mode 20 MCF7 cells (follow-up)
TABLE 1 qualitative lipid information in positive ion mode 20 MCF7 cells (follow-up)
TABLE 2 qualitative lipid information in negative ion mode 20 MCF7 cells
TABLE 2 negative ion Pattern qualitative lipid information in 20 MCF7 cells (follow-up)
TABLE 2 negative ion Pattern qualitative lipid information in 20 MCF7 cells (follow-up)
TABLE 2 negative ion Pattern qualitative lipid information in 20 MCF7 cells (follow-up)
TABLE 2 negative ion Pattern qualitative lipid information in 20 MCF7 cells (follow-up)
TABLE 2 negative ion Pattern qualitative lipid information in 20 MCF7 cells (follow-up)
Claims (8)
1. A small amount of cell lipidomic analysis method is a small amount of cell lipidomic high-sensitivity and high-flux analysis method based on a microprobe sampling-multichannel chip nano-spraying-high-resolution mass spectrometry technology, and is characterized in that:
(1) Sucking 15-25 small cells under the action of negative pressure by using a capillary microprobe under a microscope by means of a three-dimensional micro-movement operation platform;
(2) Injecting 15-25 small amount of cells into 96-384 well plate via injection pump, adding 10-20 μl of spray solvent, standing at 0-4deg.C for 0.5-1 min, and extracting lipid;
(3) The lipid extraction sample placed in the pore plate directly enters a high-resolution mass spectrum for analysis through an automatic sampler of a multichannel chip nano-spray ion source Advion NanoMate, and the first-stage mass spectrum information is acquired by adopting a spliced sectional scanning mode;
Repeating the above process for 6-10 times, and respectively carrying out peak matching on the data to obtain a characteristic ion total peak table containing mass-to-charge ratio m/z and peak intensity; subtracting the characteristic ions with solvent blank, signal-to-noise ratio less than 10 and frequency of occurrence lower than 80% in 6-10 repetitions to obtain a stable characteristic ion list, matching the first-order m/z in the sample with the accurate m/z in LIPID MAPS database, setting the quality precision to be +/-3 ppm, and obtaining a qualitative lipid list in 15-25 small cells;
In the step (2), 15-25 drawn animal cells are injected into an orifice plate through a syringe pump, and 10-20 mu L of spray solvent is added for lipid extraction, wherein the composition of the positive ion mode is chloroform: methanol: isopropyl=1:2:4, v/v/v, with 5mM ammonium formate and 0.2% formic acid as modifiers; the composition of the negative ion type lithium ion battery is chloroform: methanol: isopropanol=2:3:5, v/v/v, with 5mM ammonium acetate as modifier; the spray solvent in positive and negative ion mode contains 12 lipid internal standards including lysophosphatidylcholine LPC 12:0, phosphatidylcholine PC 15:0/15:0, phosphatidylethanolamine PE 15:0/15:0, phosphatidylglycerol PG 15:0/15:0, glycerophosphate PA 17:0/17:0, phosphatidylserine PS 16:0/16:0-d62, ceramide Cer 18:1/17:0, sphingomyelin SM d18:1/12:0, diglyceride DG 12:0/12:0, triglyceride TG 15:0/15:0, cholesterol ester ChoE 17:0, free fatty acid FFA 18:0-d3, and the internal standard concentration range is 0.01-0.05 μg/ml; standing the mixed solution at 0-4deg.C for 0.5-1 min to complete lipid extraction;
In the step (3), the lipid extraction sample directly enters a high-resolution mass spectrum for analysis through an automatic sampler of a multichannel chip nano-spray ion source, and first-stage mass spectrum information is acquired by adopting a spliced type sectional scanning mode; the operating conditions of the multi-channel chip nano-spray ion source-high resolution mass spectrum are as follows:
3.1 Multi-channel chip nano-spray ion source conditions: d-shaped chip with nozzle inner diameter of 4.1 μm; the spray voltage of positive ion mode is +1.5 kV, and the spray voltage of negative ion mode is-1.8 kV; the air pressure is 0.6 psi; the temperature of the sample injection chamber is 4 ℃; the sample volume was 5 μl;
3.2 High resolution combined quaternary rod-orbitrap mass spectra Thermo Scientific, Q Exactive-HF conditions: the temperature of the ion transmission tube is 275 ℃; the first-level m/z spliced segmented scanning windows in the positive ion mode are respectively set to be 290-390、380-480、470-570、560-610、600-650、640-690、680-730、720-770、760-810、800-850、840-940、930-1030、1020-1120、1110-1210、1200-1300 Da;, and the first-level m/z spliced segmented scanning windows in the negative ion mode are respectively set to be 150-250、240-340、330-430、420-520、510-610、600-650、640-690、680-730、720-770、760-810、800-850、840-940、930-1030、1020-1120、1110-1210、1200-1300 Da; micro-scans to be 3; the resolution is 240K; the injection time was set to 200 ms; the dynamic gain control was 1e 6 and the acquisition time was 0.6 min.
2. The method of claim 1, wherein a database of constant cell lipids is established and the reliability of the characterization of the cell lipids is verified: adding 1-2 mL lipid extraction solvent into macrocell 1×10 6-5×106, shaking, standing, centrifuging to obtain supernatant, directly feeding into high-resolution mass spectrum for analysis, collecting primary mass spectrum information by adopting a spliced segment scanning mode, and repeatedly analyzing the sample for 6-10 times; carrying out peak matching on the data to obtain a characteristic ion total peak table containing mass-to-charge ratio m/z and peak intensity; after subtracting the characteristic ions with solvent blank, signal-to-noise ratio less than 10 and occurrence frequency lower than 80% in 6-10 repetitions, obtaining a stable characteristic ion list, matching the characteristic ion list with accurate m/z in LIPID MAPS databases, setting mass accuracy to be +/-3 ppm, and taking the matched m/z peak list as a primary m/z list to carry out secondary mass spectrum acquisition of a parallel reaction monitoring mode PRM, thereby obtaining a lipid database of constant cells, wherein the lipid database comprises m/z and/or MS/MS fragment information; if the qualitative lipid of a small number of cells can be traced back to the constant cell lipid database, the qualitative method which depends on the accurate m/z in the first-order m/z matching LIPID MAPS database of a small number of cells is considered to be reliable, and the subsequent research does not need to culture the constant cells for verification.
3. The method of claim 1, wherein in step (1), the capillary microprobe is drawn by using a needle drawing instrument P-1000,Sutter Instrument, novato, and fixed on a moving slide rail connected to a three-dimensional micro-moving operation platform, the capillary microprobe is precisely moved to above the target cells by a microscope, 15-25 animal cells are sucked under the action of negative pressure, and the sampling process takes 0.4-0.6 min.
4. The method of claim 1, wherein in step (3), primary raw data is derived by Xcalibur software Thermo FISHER SCIENTIFIC, U.S.A., and peak matching is performed on the data to obtain a characteristic ion total peak table containing m/z and peak intensity; after subtracting the characteristic ions with solvent blank, signal-to-noise ratio less than 10 and frequency of occurrence lower than 80% in 6-10 repetitions, obtaining a stable characteristic ion list, matching with the accurate m/z in LIPID MAPS database, setting quality precision as +/-3 ppm, and obtaining a qualitative lipid list.
5. The method of claim 2, wherein 1-2 mL lipid extraction solvents are added to the cell culture dish in a positive ion mode consisting of chloroform: methanol: isopropanol=1:2:4, v/v/v, 5mM ammonium formate and 0.2% formic acid final volume concentration as modifier; the composition of the negative ion type lithium ion battery is chloroform: methanol: isopropanol=2:3:5, v/v/v, containing 5mM ammonium acetate as modifier; the lipid extraction solvent in positive and negative ion modes contains 12 lipid internal standards: comprises LPC 12:0、PC 15:0/15:0、PE 15:0/15:0、PG 15:0/15:0、PA 17:0/17:0、PS 16:0/16:0-d62、Cer d18:1/17:0、SM d18:1/12:0、TG 15:0/15:0/15:0、DG 12:0/12:0、ChoE 17:0、FFA 18:0-d3, with internal standard concentration of 0.01-0.05 μg/ml; extracting lipid from 1×10 6-5×106 constant cells, vibrating 1: 1 min, standing at 4deg.C for 10: 10min, centrifuging at 4deg.C at 14,000 g, collecting supernatant, directly introducing lipid extract into Q Exactive-HF mass spectrum via an automatic sampler of multi-channel chip nano-spray ion source, analyzing, collecting primary mass spectrum information by adopting a spliced segmented scanning mode, and repeatedly analyzing the sample for 6-10 times under the same operation conditions as 3.1) and 3.2).
6. The method of claim 2, wherein primary raw data is derived via Xcalibur, and peak matching is performed on the data to obtain a characteristic ion total peak table comprising m/z and peak intensity; after subtracting the characteristic ions with solvent blank, signal-to-noise ratio less than 10 and occurrence frequency less than 80% in 6-10 repetitions, obtaining a stable characteristic ion list, matching the characteristic ion list with the accurate m/z in LIPID MAPS databases, setting the mass accuracy to be +/-3 ppm, and taking the matched m/z as a list to perform PRM secondary mass spectrum acquisition; the operating conditions were as follows:
(1) Multichannel chip nano-spray ion source conditions: d-shaped chip with nozzle inner diameter of 4.1 μm; the spray voltage of positive ion mode is +1.5 kV, and the spray voltage of negative ion mode is-1.8 kV; the air pressure is 0.6 psi; the temperature of the sample injection chamber is 4 ℃; the sample volume was 5 μl;
(2) High resolution combined quaternary rod-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; the resolution is 120K; the injection time was 200 ms; the dynamic gain is controlled to be 5e 5, and the isolation window is 0.4 m/z; the collision energy is 10 eV, 20 eV, 25 eV, 30 eV, 40 eV;
checking characteristic ions, neutral loss and/or fatty acyl ion fragments in the secondary mass spectrogram one by one, and carrying out detailed structural annotation on detected lipid compounds to obtain a lipid database of constant cells, wherein lipids with fragment information are characterized by using MS and MS/MS; otherwise, only MS is used for characterization.
7. The method according to claim 2, wherein the m/z of the qualitative lipids of the small cells matches the exact m/z of the constant cell lipid database with a quality accuracy of ± 3 ppm, and the qualitative rating is Level 1 if it matches the lipids containing MS, MS/MS information in the constant cell lipid database; if the lipid can be matched with the lipid containing MS information in the constant cell lipid database, the qualitative grade is Level 2; if it cannot match the lipids in the macrocell lipid database, the qualitative rating is Level 3; if the lipid with the qualitative grade of Level 3 accounts for less than 10% of all qualitative lipids, the qualitative method which only depends on a small amount of cells to be matched with accurate m/z in LIPID MAPS database is considered to be reliable, and the follow-up study does not need to depend on constant cells to assist in qualitative, so that the method can be directly used for metabonomics analysis of rare cells.
8. The method of claim 1 or 2, wherein the cell is a human or mammalian cell.
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