CN114609225A - Small cell lipidomics analysis method - Google Patents
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Abstract
The invention discloses a high-sensitivity and high-flux lipidomics analysis method aiming at a small number of cells (15-25) by adopting a microprobe sampling-multichannel chip nano-jet-high resolution mass spectrometry technology. The method adopts a capillary microprobe to accurately sample 15-25 animal cells under a microscope, directly extracts lipid in a pore plate, directly enters a high-resolution mass spectrum for analysis through a multichannel chip nano-jet ion source, collects primary mass spectrum information by adopting a splicing type segmented scanning mode, and obtains qualitative lipid after database matching. The invention realizes that the single analysis only needs 2 minutes of flux, more than 500 lipids can be identified from 20 breast cancer cells, has the characteristics of simple process, high sensitivity and high analysis flux, and is suitable for the high-sensitivity and high-flux analysis of rare cells which are difficult to obtain, such as stem cells and circulating tumor cell lipidosome.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a high-sensitivity and high-flux lipidomics analysis method aiming at a small amount of cells and adopting a microprobe sampling-multichannel chip nano-jet-high resolution mass spectrometry technology.
Background
Metabonomics is the systematic study of the response of metabolites in vivo in a multivariate dynamic state when a living body is subjected to intrinsic gene mutation, pathophysiological change or external environmental stimulus. The cells are basic units of the structure and the function of a living body, and the metabonomics research taking the cells as objects is helpful for revealing the process and the rule of life activities. Conventional metabolomics studies in order to increase the number of quantifiable metabolites in a cell extract, metabolomic analysis is usually performed using a large number of cells (e.g., millions of cells). However, reducing the number of cells required for metabolomics would be advantageous for many studies, such as stem cells, circulating tumor cells, and primary cells obtained from tissues, which are rare and difficult to obtain; on the other hand, if the metabonomics research of a small amount of cells is feasible, a large amount of cells do not need to be cultured, so that the manpower and material resources can be saved, the test period is shortened, the experiment cost is greatly reduced, and the cell biology research is more convenient. Therefore, it is of great interest to develop metabolomic analysis methods for small numbers of cells.
The challenge of small-scale cellular metabolomics analysis is the highly sensitive and high-throughput detection of metabolites. The liquid chromatography-mass spectrometry (LC-MS) technology has become the mainstream technology of current metabonomics analysis and research due to its characteristics of high sensitivity, strong specificity, diversification, etc. However, LC chromatographic separation prior to mass spectrometric detection is often time consuming and difficult to achieve for high throughput analysis. In addition, conventional electrospray ionization sources (ESI) generally have strong matrix interference and ion suppression effects, and cannot meet the sensitivity requirements of small-amount cell metabolite analysis. On the other hand, the acquisition of a small number of cells in the currently reported small-number cellular metabonomics research still needs to be obtained by constant cell dilution counting, which is very unfavorable for the research of rare cells which are difficult to be cultured on a large scale, and the complicated and time-consuming sample pretreatment steps such as cell lysis, metabolite extraction and the like not only cause the loss of the sample, but also limit the improvement of the analysis flux.
Aiming at the problems existing in the current research of a small amount of cell metabonomics, the invention adopts the microprobe to accurately sample 15-25 cells, and directly adds the spray solvent into the pore plate to extract the lipid without complex sample pretreatment, thereby avoiding the sample loss; the directly-fed nano-liter electrospray ionization source has very high analysis flux while reducing matrix effect and improving detection sensitivity; the high-resolution mass spectrum splicing type segmented scanning mode not only can obviously improve the detection sensitivity, but also can keep higher quality precision, and greatly improves the accuracy of metabolite qualification.
Disclosure of Invention
In order to realize the analysis of a small amount of cell metabonomics, the invention establishes a high-sensitivity and high-throughput analysis method based on a microprobe sampling-multichannel chip nano-jet-high resolution mass spectrometry technology. The method has the advantages of accurate cell sampling, no need of sample pretreatment, high sensitivity, high flux and the like.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
(1) drawing a capillary microprobe by using a needle drawing Instrument (P-1000, letter Instrument, Novato, U.S. A.) and fixing the capillary microprobe on a movable slide rail connected with a three-dimensional micromotion operation platform, accurately 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 consuming 0.4-0.6min in the sampling process;
(2) injecting the 15-25 animal cells into 96-384 well plate via syringe pump, and adding 10-20 μ L spray solvent to perform lipid extraction, wherein the composition in positive ion mode is chloroform: methanol: isopropanol (1:2:4, v/v/v), 5mM ammonium formate and 0.2% formic acid as modifiers; the composition in negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v), 5mM ammonium acetate as modifier; both 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 at a concentration of 0.01-0.05. mu.g/ml. Standing the mixed solution at 0-4 deg.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 sample injector of a multichannel chip nano-jet ion source, and first-level mass spectrum information is acquired in a spliced segmented scanning mode. The operating conditions were as follows:
the conditions of the multichannel chip nano-spray ion source are as follows: d type chip (nozzle inner diameter 4.1 μm); the spray voltage in the positive ion mode is +1.5kV, and the spray voltage in the negative ion mode is-1.8 kV; air pressure of 0.6 psi; the temperature of the sample injection chamber is 4 ℃; the sample injection volume is 5 mu L;
(ii) high-resolution combined quadrupole-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; the primary m/z spliced segmented scanning window in the positive ion mode is set as 290-; micro-scan is set to 3; the resolution is 240K; the injection time was set to 200 ms; dynamic gain control to 1e6The collection time is 0.6 min;
(4) repeating the process (1-3) for 6-10 times; and (3) exporting primary mass spectrum data by using Xcalibur software, and carrying out peak matching on the data to obtain a total peak table of the characteristic ions containing m/z and peak intensity. Deducting the solvent blank, the signal-to-noise ratio is less than 10, and the characteristic ions with the frequency lower than 80% appear in 6-10 times of repetition to obtain a stably existing characteristic ion list, matching the characteristic ion list with accurate m/z in a Lipid Maps database, and setting the mass accuracy to be +/-3 ppm to obtain a qualitative Lipid list;
(5) to a constant cell (1X 10)6-5×106) 1-2mL of lipid extraction solvent was added to the petri dish, which in positive ion mode consisted of chloroform: methanol: isopropanol (1:2:4, v/v/v), with 5mM ammonium formate and 0.2% formic acid as modifiers; the anion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v), with 5mM ammonium acetate as modifier. Both of the above two extraction solventsContains 12 internal lipid 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, and the concentration of the internal standard is 0.01-0.05 mug/ml. The supernatant of the mixed solution after oscillation, standing and centrifugation is the cell lipid extract, which directly enters a high-resolution mass spectrum for analysis through an automatic sample injector of a multichannel chip nano-spray ion source, and the first-stage mass spectrum information is acquired by adopting a spliced segmented scanning mode. The samples were analyzed 6-10 times in duplicate, operating under the same conditions as in (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. Deducting the solvent blank, the signal-to-noise ratio is less than 10, and the characteristic ions with the frequency lower than 80% appear in 6-10 times of repetition to obtain a stably existing characteristic ion list, matching the characteristic ion list with accurate m/z in a Lipid Maps database, setting the mass accuracy to be +/-3 ppm, and performing PRM secondary mass spectrum acquisition by taking the matched m/z as a Lipid list. The operating conditions were as follows:
firstly, the conditions of the multi-channel chip nano-spray ion source are the same as those of the multi-channel chip nano-spray ion source (3);
(ii) high-resolution combined quadrupole-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; the resolution is 120K; the injection time is 200 ms; dynamic gain control of 5e5The isolation window is 0.4 m/z; collision energy is 10eV, 20eV, 25eV, 30eV, 40 eV;
and (3) checking characteristic ions, neutral loss and/or fatty acyl ion fragments in the secondary mass spectrogram one by one, and carrying out detailed structure annotation on the lipid, thereby obtaining a constant cell lipid database comprising MS and/or MS/MS information. Lipids with fragment information were characterized using MS, MS/MS; otherwise, characterisation was performed with MS only.
(7) Matching m/z of a small amount of cell qualitative lipid with accurate m/z in a constant cell lipid database, setting 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 Level is Level 2; if the lipid cannot be matched with the lipid in the constant cell lipid database, the qualitative grade is Level 3. If the proportion of Lipid with qualitative grade of Level 3 in all qualitative lipids is less than 10%, the qualitative method of matching accurate m/z in a Lipid Maps database only depending on a small number of cells and the first-Level m/z is reliable, and subsequent research does not need to depend on constant cell for auxiliary qualification and can be directly used for metabonomics analysis of rare cells.
The invention realizes that the single analysis only needs 2 minutes of flux, more than 500 lipids can be identified from 20 breast cancer cells, has the characteristics of simple process, high sensitivity and high analysis flux, and is suitable for the high-sensitivity and high-flux analysis of rare cells which are difficult to obtain, such as stem cells and circulating tumor cell lipidosome.
The invention adopts the microprobe to accurately sample 15-25 cells, and directly adds the spray solvent into the pore plate to complete lipid extraction, so that the sample pretreatment is not needed, and the sample loss is avoided as much as possible; the directly-fed nanoliter electrospray ionization source has very high analysis flux while reducing matrix effect and improving detection sensitivity; the high-resolution mass spectrum splicing type segmented scanning mode can further improve the detection sensitivity, can also keep higher quality precision, and greatly improves the accuracy of metabolite qualification.
Drawings
FIG. 1(A) lipid characterization strategy for small numbers of cells; (B) the construction process of the constant cell lipid database is shown in the figure.
FIG. 2 is a schematic diagram of an m/z mass window setup for a tiled segmented scan.
Figure 3 percentage of different classes of lipids qualitative by small number (20) of cells in positive and negative ion mode.
Figure is a schematic representation of the results of validation of qualitative lipids of 420 MCF7 cells in the constant cell lipid database.
FIG. 5 Main component analysis score chart of lipids with p <0.05 in three cancer cells, MCF7, MHCC97H and VCaP.
Detailed Description
Embodiments of the invention are described in detail below with reference to the attached table drawings: the embodiments are carried out on the premise of the technical scheme of the invention, and detailed embodiments and specific operation processes are given, but the scope of the invention is not limited to the following embodiments.
Example one
The flow chart established by the method for high-sensitivity and high-throughput analysis of small-amount cell lipidomics based on the microprobe sampling-multichannel chip nano-jet-high resolution mass spectrometry technology is shown in figure 1, and the specific implementation steps are as follows:
(1)20 MCF7 cells were sampled:
drawing a capillary microprobe (the inner diameter of a needle point is 5-10 mu m) 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, accurately moving the capillary microprobe above a target cell by virtue of a microscope, sucking 20 breast cancer cells (MCF7) under the action of negative pressure, and repeatedly sampling for 6 times;
(2) lipid extraction:
the 20 MCF7 cells aspirated were injected into a 96-well plate via a syringe pump, and 15 μ L of spray solvent was added for lipid extraction, which consisted of chloroform in positive ion mode: methanol: isopropanol (1:2:4, v/v/v) containing 5mM ammonium formate and a final volume concentration of 0.2% formic acid as modifier; the composition in negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v) containing 5mM ammonium acetate as modifier; the two spray 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, FFA18:0-d3, and the concentration of the internal standard is 0.01-0.05 mug/ml. Standing the mixed solution at 4 deg.C for 1min to complete lipid extraction;
(3) direct sample introduction-acquisition of high-resolution mass spectrum:
the lipid extract sample directly enters a high-resolution mass spectrum for analysis through an automatic sample injector of a multichannel chip nano-jet ion source, and first-level mass spectrum information is acquired in a spliced segmented scanning mode. The m/z mass window segmented scan setup is shown in fig. 2.
The conditions of the multichannel chip nano-spray ion source are as follows: d type chip (nozzle inner diameter 4.1 μm); the spraying voltage under the positive ion mode is +1.5kV, and the spraying voltage under the negative ion mode is-1.8 kV; air pressure of 0.6 psi; the temperature of the sample introduction chamber is 4 ℃; the sample injection volume is 5 mu L;
(ii) high resolution combined quadrupole-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; the mass window of the spliced segmented scanning in the positive ion mode is set as 290-; the micro-scan is set to 3; the resolution is 240K; the injection time was set to 200 ms; dynamic gain control of 1e6The collection time is 0.6 min;
(4) lipid characterization:
and (3) exporting primary mass spectrum data by using Xcalibur software, and carrying out peak matching on the data to obtain a total peak table of the characteristic ions containing m/z and peak intensity. Deducting the solvent blank, the signal-to-noise ratio is less than 10, and the characteristic ions with the frequency lower than 80% appear in 6 times of repetition to obtain a stably existing characteristic ion list, matching the stably existing characteristic ion list with the accurate m/z in a Lipid Maps database, setting the mass accuracy to be +/-3 ppm, and obtaining the qualitative Lipid, which is shown in tables 1 and 2 and fig. 3;
(5) constant (5X 10)6) MCF7 cell lipid extraction:
5×106MCF7 cell samples were placed in a petri dish, the culture solution was removed sequentially, washed three times with PBS, and 1ml of a lipid extraction solvent consisting of chloroform in positive ion mode: methanol: isopropanol (1:2:4, v/v/v) containing 5mM ammonium formate and 0.2% formic acid as modifier; the composition in negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v) containing 5mM ammonium acetate as modifier; both extraction 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, with an internal standard concentration of 0.01-0.05. mu.g/ml. Transferring the mixed solution into a 2ml EP tube, vortexing for 10s, shaking for 10min, standing at 4 ℃ for 10min, centrifuging for 10min under the condition of 14000g, and taking 800 μ L of supernatant (namely lipid extract of macrocells) to be subjected to sample injection analysis;
(6) constant MCF7 cells were directly injected-first order acquisition of high resolution mass spectra:
a constant cell lipid extract sample directly enters a high-resolution mass spectrum for analysis through an automatic sample injector of a multichannel chip nano-spray ion source, primary mass spectrum information is acquired in a spliced segmented scanning mode, the sample is repeatedly analyzed for 6 times, and the operation conditions are the same as those in the step (3);
(7) direct sample injection of constant MCF7 cells-high resolution mass spectrometry secondary acquisition:
and (4) exporting the primary mass spectrum data in the step (6) through an Xcaliibur software, and performing peak matching on the data by adopting an autonomously written Python program to obtain a characteristic ion total peak table containing m/z and peak intensity. Deducting the solvent blank, the signal-to-noise ratio is less than 10, and the characteristic ions with the frequency lower than 80% appear in 6 times of repetition, obtaining a stably existing characteristic ion list, matching the characteristic ion list with accurate m/z in a Lipid Maps database, setting the mass precision to be +/-3 ppm, and performing PRM secondary mass spectrum acquisition by taking the matched characteristic ions as a Lipid list. The operating conditions of the multi-channel chip nano-jet ion source-high resolution mass spectrum are as follows:
firstly, the conditions of the multi-channel chip nano-spray ion source are the same as those of (3);
(ii) high-resolution combined quadrupole-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; the resolution is 120K; the injection time is 200 ms; dynamic gain control to 5e5The isolation window is 0.4 m/z; collision energy is 10eV, 20eV, 25eV, 30eV, 40 eV;
(8) establishment of lipid database of MCF7 cells in constant quantity:
and (3) checking characteristic ions, neutral loss and/or fatty acyl ion fragments in the secondary mass spectrogram one by one, and carrying out detailed structure annotation on the lipid, thereby obtaining a constant cell lipid database comprising MS and/or MS/MS information. Lipids with fragment information were characterized using MS, MS/MS; otherwise, characterisation was performed with MS only.
(9) The qualitative lipids of 20 MCF7 cells were validated based on the constant cell lipid database:
the m/z of the 20 MCF7 cell qualitative lipids matches the accurate m/z of the constant MCF7 cell lipid database, the set mass precision is +/-3 ppm, and if the mass precision can be matched with the lipids 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 Level is Level 2; if the lipid can not be matched with the lipid in the constant cell lipid database, the qualitative grade is Level 3, and the verification result is shown in figure 4.
Example two
Typing of different cancer cells (breast cancer cell MCF7, liver cancer cell MHCC97H, prostate cancer cell VCaP):
(1) sampling of 20 different cancer cells:
drawing a capillary microprobe (the inner diameter of a needle point is 5-10 mu m) by using a needle drawing Instrument (P-1000, Sun Instrument, Novato, U.S. A.) and fixing the capillary microprobe on a movable slide rail connected with a three-dimensional micro-movable operation platform, accurately moving the capillary microprobe to the upper part of a target cell by virtue 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:
injecting 20 MCF7, MHCC97H and VCaP cells which are respectively sucked into a 96-well plate through a syringe pump, and adding 15 mu L of spray solvent for lipid extraction, wherein the components of the solution in a positive ion mode are chloroform: methanol: isopropanol (1:2:4, v/v/v) containing 5mM ammonium formate and 0.2% formic acid as modifier; the composition in negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v) containing 5mM ammonium acetate as modifier; the two spray 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, FFA18:0-d3, and the concentration of the internal standard is 0.01-0.05 mu g/ml. Standing the mixed solution at 4 deg.C for 1min to complete lipid extraction;
(3) direct sample introduction-high resolution mass spectrum acquisition:
the lipid extract sample directly enters a high-resolution mass spectrum for analysis through an automatic sample injector of a multichannel chip nano-spray ion source, and a spliced segmented scanning mode is adopted to acquire primary mass spectrum information.
The conditions of the multichannel chip nano-spray ion source are as follows: d type chip (nozzle inner diameter 4.1 μm); the spraying voltage under the positive ion mode is +1.5kV, and the spraying voltage under the negative ion mode is-1.8 kV; air pressure of 0.6 psi; the temperature of the sample injection chamber is 4 ℃; the sample injection volume is 5 mu L;
(ii) high-resolution combined quadrupole-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; the mass window of the spliced segmented scan in the positive ion mode is set as 290-; the micro-scan is set to 3; the resolution is 240K; the injection time was set to 200 ms; dynamic gain control to 1e6The collection time is 0.6 min;
(4) lipid characterization:
and (3) exporting primary mass spectrum data by using Xcalibur software, and carrying out peak matching on the data to obtain a total peak table of the characteristic ions containing m/z and peak intensity. Deducting the solvent blank, the signal-to-noise ratio is less than 10, and the characteristic ions with the frequency lower than 80% appear in 10 times of repetition to obtain a stably existing characteristic ion list, matching the stably existing characteristic ion list with the accurate m/z in a Lipid Maps database, setting the mass accuracy to be +/-3 ppm, and respectively obtaining the qualitative 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 a sample, Student's t test is used for significance analysis, and lipid with p <0.05 is selected for main component analysis (figure 5), so that different cancer cells are effectively distinguished, and the composition and relative content of the lipid of different cancer cells are obviously different.
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 20 MCF7 cells in Positive ion mode
TABLE 1 qualitative lipid information in 20 MCF7 cells in Positive ion mode (continuous)
TABLE 1 qualitative lipid information in 20 MCF7 cells in Positive ion mode (continuous)
TABLE 1 qualitative lipid information in 20 MCF7 cells in Positive ion mode (continuous)
TABLE 1 qualitative lipid information in 20 MCF7 cells in Positive ion mode (continuous)
TABLE 1 qualitative lipid information in 20 MCF7 cells in Positive ion mode (continuous)
TABLE 1. qualitative lipid information in 20 MCF7 cells in Positive ion mode (continuous)
TABLE 1 qualitative lipid information in 20 MCF7 cells in Positive ion mode (continuous)
TABLE 1 qualitative lipid information in 20 MCF7 cells in Positive ion mode (continuous)
TABLE 2 qualitative lipid information in negative ion mode 20 MCF7 cells
TABLE 2 qualitative lipid information in negative ion mode 20 MCF7 cells (continuous)
TABLE 2 qualitative lipid information in negative ion mode 20 MCF7 cells (continuous)
TABLE 2 qualitative lipid information in negative ion mode 20 MCF7 cells (continuous)
TABLE 2. qualitative lipid information in negative ion mode 20 MCF7 cells (continuous)
TABLE 2. qualitative lipid information in negative ion mode 20 MCF7 cells (continuous)
Claims (10)
1. A small amount of cell lipidomics analysis method is a high-sensitivity and high-flux analysis method of small amount of cell lipidomics based on microprobe sampling-multichannel chip nano-jet-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 virtue of a three-dimensional micro-moving operation platform;
(2) injecting 15-25 small amount of cells into 96-384 well plate via injection pump, adding 10-20 μ L spray solvent, standing at 0-4 deg.C for 0.5-1min, and extracting lipid;
(3) a lipid extraction sample placed in a pore plate directly enters a high-resolution mass spectrum for analysis by an automatic sample injector of a multichannel chip nano-spray ion source (Advion NanoMate), and primary mass spectrum information is acquired by adopting a spliced segmented scanning mode;
repeating the 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; deducting the solvent blank, the signal-to-noise ratio is less than 10, and the characteristic ions with the frequency lower than 80% appear in 6-10 times of repetition to obtain a stably existing characteristic ion list, matching the first-order m/z in the sample with the accurate m/z in the Lipid Maps database, setting the mass precision to be +/-3 ppm, and obtaining a qualitative Lipid list in 15-25 small cells.
2. The method of claim 1, wherein a constant cell lipid database is establishedAnd verifying the reliability of the cell lipid characterization: constant cell (1X 10)6-5×106) Adding 1-2mL of lipid extraction solvent, shaking, standing, centrifuging, collecting supernatant, directly entering high-resolution mass spectrometry for analysis, collecting primary mass spectrometry information by adopting a spliced segmented scanning mode, and repeatedly analyzing a 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; deducting solvent blank, enabling the signal-to-noise ratio to be less than 10 and enabling the occurrence frequency of characteristic ions to be lower than 80% in 6-10 times of repetition to obtain a stably existing characteristic ion list, matching the characteristic ion list with accurate m/z in a Lipid Maps database, setting the mass accuracy to be +/-3 ppm, taking the matched m/z peak list as a primary m/z list to carry out secondary mass spectrum acquisition in a parallel reaction monitoring mode (PRM), and thus obtaining a Lipid database of constant cells, wherein the Lipid database comprises m/z and/or MS/MS fragment information; if a small amount of cell qualitative Lipid can be traced back to a constant cell Lipid database, the qualitative method of matching the accurate m/z in the Lipid Maps database by only relying on the first-order m/z of a small amount 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, U.S. A.) and fixed on a movable slide rail connected with a three-dimensional micro-moving operation platform, the capillary microprobe is precisely moved above the target cell by means of 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 according to claim 1, wherein 15 to 25 animal cells aspirated in the step (2) are injected into a well plate via a syringe pump, and 10 to 20 μ L of a spray solvent is added to perform lipid extraction, which has a composition of chloroform: methanol: isopropanol (1:2:4, v/v/v), with 5mM ammonium formate and 0.2% formic acid as modifiers; the composition in the negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v), with 5mM ammonium acetate as modifier; the spray solvent under the 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, glycerophosphatidic acid (PA)17:0/17:0, Phosphatidylserine (PS)16:0/16:0-d62, ceramide (Cer) d18:1/17:0, Sphingomyelin (SM) d18:1/12:0, Diglyceride (DG)12:0/12:0, Triglyceride (TG)15:0/15:0/15:0, cholesterol ester (ChoE)17:0, Free Fatty Acid (FFA)18:0-d3, and the concentration range of the internal standard is 0.01-0.05 mug/ml; standing the mixed solution at 0-4 deg.C for 0.5-1min to complete lipid extraction.
5. The method according to claim 1 or 2, wherein in the step (3), the lipid extracted sample directly enters the high-resolution mass spectrum for analysis through an autosampler of the multichannel chip nano-jet ion source, and the first-order mass spectrum information is acquired by adopting a spliced segmented scanning mode; the operating conditions of the multi-channel chip nano-jet ion source-high resolution mass spectrum are as follows:
(1) the conditions of the multi-channel chip nano-spray ion source are as follows: d type chip (nozzle inner diameter 4.1 μm); the spraying voltage of the positive ion mode is +1.5kV, and the spraying voltage of the negative ion mode is-1.8 kV; air pressure of 0.6 psi; the temperature of the sample injection chamber is 4 ℃; the sample volume was 5 μ L;
(2) high resolution combined quadrupole-orbitrap mass spectrometry (Thermo Scientific, Q active-HF) conditions: the temperature of the ion transmission tube is 275 ℃; the primary m/z spliced segmented scanning windows in the positive ion mode are respectively set to 290-; the primary m/z spliced segmented scanning windows in the negative ion mode are respectively 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; the micro-scan is set to 3; the resolution is 240K; the injection time was set to 200 ms; dynamic gain control to 1e6The collection time is 0.6 min.
6. The method according to claim 1, wherein in step (3), primary raw data is derived through 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; deducting the solvent blank, the signal-to-noise ratio is less than 10, and the characteristic ions with the frequency lower than 80% appear in 6-10 times of repetition to obtain a stably existing characteristic ion list, matching the characteristic ion list with the accurate m/z in a Lipid Maps database, and setting the mass accuracy to be +/-3 ppm to obtain a qualitative Lipid list.
7. The method of claim 2, wherein 1-2mL of lipid extraction solvent is added to the cell culture dish, and the composition in positive ion mode is chloroform: methanol: isopropanol (1:2:4, v/v/v) containing 5mM ammonium formate and a final volume concentration of 0.2% formic acid as modifier; the composition in the negative ion mode is chloroform: methanol: isopropanol (2:3:5, v/v/v) containing 5mM ammonium acetate as modifier; the lipid extraction solvents in the positive and negative ion mode all contain 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 and FFA18:0-d3, and the concentration of the internal standard is 0.01-0.05 mug/ml; for constant number of cells (1X 10)6-5×106) Extracting lipid, shaking for 1min, standing at 4 deg.C for 10min, centrifuging at 4 deg.C at 14,000g, collecting supernatant, directly introducing the lipid extract into Q active-HF mass spectrometer via automatic sampler of multichannel chip nano-ion spray source for analysis, collecting primary mass spectrum information by using spliced segmented scanning mode, and repeatedly analyzing the sample for 6-10 times under the same operation conditions as in claim 5.
8. The method according to claim 2, characterized in that primary raw data is derived through Xcalibur, and peak matching is performed on the data to obtain a characteristic ion total peak table containing m/z and peak intensity; deducting the solvent blank, the signal-to-noise ratio is less than 10, and the occurrence frequency of characteristic ions in 6-10 times of repetition is less than 80%, obtaining a stably existing characteristic ion list, matching the characteristic ion list with accurate m/z in a Lipid Maps database, setting the mass accuracy to be +/-3 ppm, and performing PRM secondary mass spectrum acquisition by taking the matched m/z as a list; the operating conditions were as follows:
(1) the conditions of the multi-channel chip nano-spray ion source are the same as the conditions in claim 5;
(2) high resolution combined quadrupole-orbitrap mass spectrometry conditions: the temperature of the ion transmission tube is 275 ℃; the resolution is 120K; the injection time is 200 ms; dynamic gain control to 5e5The isolation window is 0.4 m/z; collision energy is 10eV, 20eV, 25eV, 30eV, 40 eV;
checking the characteristic ions, neutral loss and/or fatty acyl ion fragments in the secondary mass spectrogram one by one, and carrying out detailed structure annotation on the detected lipid compound, thereby obtaining a lipid database of the constant cells, wherein the lipid with fragment information is characterized by MS and MS/MS; otherwise, characterisation was performed with MS only.
9. The method of claim 2, wherein the m/z of the small amount of cell qualitative lipid matches the exact m/z of the constant cell lipid database, the mass accuracy is set to ± 3ppm, and if it can match the lipid containing MS, MS/MS information in the constant cell lipid database, the qualitative Level is 1; if the lipid can be matched with the lipid containing MS information in the constant cell lipid database, the qualitative Level is Level 2; if the lipid can not be matched with the lipid in the constant cell lipid database, the qualitative grade is Level 3; if the proportion of the Lipid with the qualitative grade of Level 3 in all the qualitative lipids is less than 10%, the qualitative method of matching accurate m/z in the Lipid Maps database only depending on a small number of cells by one grade is reliable, and subsequent research does not need to depend on the assistance of constant cells for qualitative analysis, and can be directly used for metabonomics analysis of rare cells.
10. The method of claim 1 or 2, wherein the cell is a human or mammalian cell.
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