CN114563247A

CN114563247A - Lipidomics sample pretreatment method and detection method and device thereof

Info

Publication number: CN114563247A
Application number: CN202210202555.9A
Authority: CN
Inventors: 成晓亮; 余静; 周岳; 张伟; 郑可嘉
Original assignee: Jiangsu Pinsheng Medical Technology Group Co ltd
Current assignee: Jiangsu Pinsheng Medical Technology Group Co ltd; Nanjing Pinsheng Medical Technology Co ltd
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2022-05-31

Abstract

The application belongs to the technical field of omics analysis methods, and relates to a lipidomics sample pretreatment method, a detection method and a device thereof, wherein the method comprises the following steps: uniformly mixing an experiment sample added with an isopropanol solvent in a vortex manner under a first condition; centrifuging the test sample after vortex mixing at low temperature under a second condition to obtain supernatant of the micro sample; transferring the supernatant to a protein precipitation plate; centrifuging the supernatant in the protein precipitation plate at a low temperature under a third condition to obtain a filtrate; the filtrate was determined as a lipidomics sample. According to the technical scheme, by adopting an isopropanol single-phase extraction system, the degradation of lipid metabolites in the preparation process is reduced, meanwhile, a protein precipitation plate is added in the experimental process, the protein removal efficiency is improved, and an automatic workstation is introduced to improve the repeatability and efficiency of the experiment.

Description

Lipidomics sample pretreatment method and detection method and device thereof

Technical Field

The application relates to the technical field of omics analysis methods, in particular to a lipidomics sample pretreatment method and a detection method and device thereof.

Background

Lipidomics (Lipidomics) are a comprehensive description of lipid molecular species and their biological roles (encompassing factors such as lipid metabolism, functional protein expression, and gene regulation). The research of lipidomics is of great significance for understanding the occurrence mechanism and the process of diseases. During lipid analysis, due to sample matrix interference or analyte concentration limitations, it is often necessary to pre-treat the sample for optimal analytical performance.

In the existing lipidomics sample pretreatment method, a liquid-liquid extraction method is usually adopted for extracting the lipid. Liquid-liquid extraction methods make use of the difference in the partition coefficients of analytes in two immiscible solvents to achieve separation of different substances. Liquid-liquid extraction in the process of lipid extraction, an organic phase containing lipids is in the lower layer of a two-phase solution.

In the prior art, no matter the lower layer liquid is directly removed or the upper layer liquid is removed, the solution pollution or the sample loss can be caused, and meanwhile, due to the technical barrier of layered sampling, the prior sample pretreatment method is difficult to be applied to automatic equipment.

Disclosure of Invention

The application provides a lipidomics sample pretreatment method and a detection method and device thereof, which reduce the degradation of lipid metabolites in the preparation process by adopting an isopropanol single-phase extraction system, improve the protein removal efficiency by adding a protein precipitation plate in the experimental process, and improve the experimental reproducibility and efficiency by introducing an automatic workstation.

In a first aspect, the present application shows a method for lipidomic sample pretreatment, said method comprising: uniformly mixing an experiment sample added with an isopropanol solvent in a vortex manner under a first condition; centrifuging the test sample subjected to vortex mixing at low temperature under a second condition to obtain a supernatant of the trace sample; transferring the supernatant to a protein precipitation plate; centrifuging the supernatant in the protein precipitation plate at a low temperature under a third condition to obtain a filtrate; the filtrate was determined as a lipidomics sample. By adopting the embodiment, an isopropanol single-phase extraction system is adopted, fewer experimental steps are applied, and the degradation of lipid metabolites in the preparation process is reduced; compared with the existing extraction system, the isopropanol single-phase extraction system has lower volatility and can ensure the accuracy of supernatant transfer volume in the experimental process; the extraction solvent is compatible with the mobile phase used in analysis and detection, freeze-drying and redissolving are not needed, and the supernatant can be directly subjected to mass spectrometry, so that the degradation and loss of a long-time freeze-drying redissolved sample are avoided. In addition, due to the addition of the protein precipitation plate, the traditional link of freezing and standing the sample for 1-2 hours is replaced, the protein precipitation time is saved, the protein precipitation removal efficiency is improved, and therefore the UHPLC-MS ionization inhibition and MS source pollution are reduced, and the detection sensitivity is improved.

In some embodiments, the sample to which the isopropanol solvent is added is vortexed under a first condition comprising: the volume ratio of the experimental sample to the isopropanol solvent is 1: 3 to 1: 4. by adopting the embodiment, the isopropanol solvent and the experimental sample can be dissolved in a proper volume ratio to achieve a good single-phase extraction result.

In some embodiments, the first condition is configured to mix the test sample at 1000-. By adopting the embodiment, the experimental samples can be fully and uniformly mixed.

In some embodiments, the second condition is configured to centrifuge the test sample at 1000-. With this embodiment, the experimental sample can be centrifuged to obtain a supernatant.

In some embodiments, the third condition is configured to centrifuge the test sample at 1000-2200g for 5-10 minutes at 4-8 ℃. With this embodiment, the experimental sample can be centrifuged to obtain a filtrate.

In a second aspect, the present application also features a method of detecting a lipidomic sample, said method comprising: obtaining a lipidomic sample, which is subjected to sample pre-treatment by the method as described in the above example; and carrying out mass spectrum detection analysis on the lipidomic sample to obtain a detection result of the lipidomic sample. By adopting the embodiment, the lipidomics sample subjected to sample pretreatment can be subjected to mass spectrometry detection and analysis, and a more accurate lipidomics sample detection result can be obtained.

In a third aspect, the present application also shows a lipidomic sample pre-treatment device, said device being suitable for use in the method according to the above example.

In some embodiments, the device is an automated workstation. By adopting the embodiment, the isopropanol single-phase extraction system is added, so that the total volume of liquid in the sample pretreatment process is lower than 2ml (the maximum volume of the existing 96-pore plate is 2 ml/pore, and if the total volume of the solution is larger than 2ml, the 96-pore plate cannot be used for sample pretreatment, and therefore, an automatic workstation cannot be used for high-throughput experiment), the experimental sample solution can be transferred into the 96-pore plate (or 385-pore plate), and then the experimental sample solution is combined with the automatic workstation to perform all pretreatment operations, and the reproducibility and the efficiency of the whole process are improved.

This application has reduced the degradation of lipid metabolite in the preparation process through adopting the isopropyl alcohol single-phase extraction system, the use of protein precipitation board has been added simultaneously in the experimentation, the efficiency of getting rid of protein has been improved, and introduced automatic workstation and improved the reproducibility and the efficiency of experiment, and saved sample nitrogen and blown and redissolution link, the mobile phase that adopts when making sample solvent system and last computer detect is basically unanimous, can be compatible with the mass spectrograph, consequently can directly inject the mass spectrum and can the analysis and detection.

Drawings

In order to more clearly describe the technical solutions of the embodiments of the present application, the drawings required to be used in the embodiments are briefly described below, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

Figure 1 shows a lipidomic sample pretreatment method flow diagram according to some embodiments;

figure 2 shows a schematic of mass spectrum results for a control group and an experimental group according to some embodiments;

figure 3 shows a graph of a statistical analysis of lipid molecule number and coverage class according to some embodiments;

FIG. 4 illustrates a software schematic of an automated workstation according to some embodiments;

FIG. 5 shows an internal block diagram of an automated workstation according to some embodiments (1-thermostatic mixer shaker, 2-200 microliter tips, 3-PCR plate, 4-50 microliter tips, 5-0.5mL 96-well plate, 7-12 channels, 9-protein precipitation plate);

figure 6 shows an overall sample lipid intensity distribution ranking map according to some embodiments;

figure 7 shows a sample lipid molecule identification number histogram according to some embodiments;

figure 8 shows a statistical plot of the total intensity of lipid molecules identified for a portion of the sample, according to some embodiments.

Detailed Description

To make the objects, embodiments and advantages of the present application clearer, the following description of exemplary embodiments of the present application will clearly and completely describe the exemplary embodiments of the present application with reference to the accompanying drawings in the exemplary embodiments of the present application, and it is to be understood that the described exemplary embodiments are only a part of the embodiments of the present application, and not all of the embodiments.

All other embodiments, which can be derived by a person skilled in the art from the exemplary embodiments described herein without making any inventive step, fall within the scope of the appended claims. In addition, while the disclosure herein has been presented in terms of one or more exemplary examples, it should be appreciated that aspects of the disclosure may be implemented solely as a complete embodiment. It should be noted that the brief descriptions of the terms in the present application are only for the convenience of understanding the embodiments described below, and are not intended to limit the embodiments of the present application. These terms should be understood in their ordinary and customary meaning unless otherwise indicated.

It should be understood that the terms "first," "second," "third," and the like in the description and in the claims of the present application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used are interchangeable under appropriate circumstances and can be implemented in sequences other than those illustrated or otherwise described herein with respect to the embodiments of the application, for example.

The areas of expertise in this application are explained first.

As used herein, unless otherwise indicated, the term "sample" refers to a biological sample used in omics studies, including, but not limited to, animal samples (e.g., human or animal blood, urine, feces, saliva, hair, cells, tissue, organs, etc.), plant samples (e.g., plant roots, stems, leaves, flowers, fruits, seeds, etc.), microbial samples (e.g., microbial cells, spores, fermentation broth, culture fluid, etc.), subcellular structural samples (e.g., organelle mitochondria, exosomes, vesicles, etc.), and the like.

As used herein, unless otherwise indicated, the term "extraction solvent" refers to a solvent used to extract a desired component of a substance from a sample, "single-phase extraction solvent" refers to a single solvent used as an extraction solvent or a combination of two or more solvents in homogeneous form, "biphasic extraction solvent" refers to a combination of two or more solvents in partially or completely immiscible two phases used as an extraction solvent.

Unless otherwise indicated, the term "lipid" as used herein is an important constituent of cell membranes, and is involved in vital activities such as energy storage, signal transduction, and the like, and plays an important role in a living body.

Unless otherwise indicated, the term "lipidomics" as used herein is a comprehensive description of lipid molecular species and their biological role, encompassing factors such as lipid metabolism, functional protein expression, and gene regulation, and is of great importance in understanding the mechanisms and processes of disease development.

In the lipidomics analysis process, because the interference of a sample matrix or the concentration limit value of a sample generally needs to be subjected to sample pretreatment, in the existing sample pretreatment process, the sample extraction method is generally a liquid-liquid extraction method, and the liquid-liquid extraction method is to realize the separation of different substances by utilizing the difference of distribution coefficients according to the fact that the sample is in two mutually insoluble solvents. The liquid-liquid extraction method comprises the following steps: at least one of Floch extraction, Bligh-Dyer extraction and Matyash extraction.

Wherein the Floch extraction method adopts a two-phase extraction solvent of chloroform-methanol-water, and the volume ratio of the chloroform-methanol-water is 8:4: 3; the Bligh-Dyer extraction method adopts a two-phase extraction solvent of chloroform-methanol-water, and the volume ratio of the chloroform-methanol-water to the water is 2: 2: 1.8; to reduce toxicity, the Matyash extraction method uses a biphasic extraction solvent, methyl tert-butyl ether (MTBE), instead of chloroform. By the above extraction method, the organic phase containing the lipid is in the lower layer of the two-phase solution during the lipid extraction process. Therefore, in the conventional lipid extraction process, solution contamination or sample loss may be caused whether the lower layer liquid is directly removed or the upper layer liquid is removed, and meanwhile, the conventional sample pretreatment method is difficult to be applied to an automatic device due to the technical barrier of stratified sampling.

In order to solve the problems that the existing sample pretreatment method is low in extraction efficiency, high in degradation degree of lipid metabolites in the preparation process and incapable of being applied to automatic equipment, the application provides a lipidomics sample pretreatment method.

Fig. 1 is a schematic flow chart of a lipidomics sample pretreatment method shown in the examples of the present application. As shown in fig. 1, the method includes:

step S101, uniformly mixing the experiment sample added with the isopropanol solvent in a vortex mode under a first condition.

In some embodiments, the volume ratio of the experimental sample to the isopropanol solvent is 1: 3 to 1: 4.

in some embodiments, the first condition is configured to mix the test sample at 1000-.

In the specific implementation, taking an experimental sample as a plasma sample as an example, 100 microliters of the plasma sample is placed in a 0.5 milliliter 96-well plate and placed on a constant-temperature mixing shaker, an isopropanol solution with the volume 3 times that of the plasma sample is absorbed and added into the plasma sample, and vortex mixing is performed at the rotating speed of 1000rpm for 5 minutes to extract lipid molecules.

And S102, centrifuging the test sample after vortex mixing at low temperature under a second condition to obtain the supernatant of the micro sample.

In some embodiments, the second condition is configured to centrifuge the test sample at 1000-.

In a specific implementation, a 96-well plate on a constant temperature mixing shaker, in which a plasma sample is placed, is centrifuged at 4 ℃ and 2200g for 5 minutes.

Step S103, transferring the supernatant to a protein precipitation plate.

In the specific implementation, after the centrifugation is finished, the 96-well plate is placed back on a constant-temperature mixing shaker, and then the supernatant obtained by centrifuging the plasma sample in the 96-well plate is transferred to a protein precipitation plate.

Step S104, centrifuging the supernatant in the protein precipitation plate at a low temperature under a third condition to obtain a filtrate.

In some embodiments, the third condition is configured to centrifuge the test sample at 1000-2200g for 5-10 minutes at 4-8 ℃.

In a specific implementation, the supernatant in the protein precipitation plate was centrifuged at 2200g for 5 minutes at 4 ℃ to obtain a filtrate.

Step S105, determining the filtrate as a lipidomic sample.

In the specific implementation, the filtrate is placed on a constant-temperature mixing shaking table, 50 microliters of the filtrate is transferred and placed in a PCR plate, and the lipidomics sample is determined to wait for mass spectrometry detection and analysis.

The present application also shows a method of detecting a lipidomic sample, said method comprising:

obtaining a lipidomic sample, which is subjected to sample pre-treatment by the method in the above example; and carrying out mass spectrometric detection analysis on the lipidomic sample to obtain a detection result of the lipidomic sample.

In specific implementation, the technical scheme shown in the application is used as an experimental group, and a first control group, a second control group and a third control group are arranged to compare the detection results of lipidomic samples. Wherein, the experimental samples in the experimental group, the first control group, the second control group and the third control group are set as plasma samples.

In some embodiments, the first control group extraction architecture is configured to: MTBE-methanol-water extraction system.

The detection process comprises the following steps: placing 200 microliters of the plasma sample into a 10 milliliter centrifuge tube, adding 1000 microliters of methanol solvent, uniformly mixing by vortex, adding 3 milliliters of MTBE solvent, oscillating for 5 minutes by vortex, adding 1000 microliters of water, and vortexing for 30 minutes at room temperature. Followed by standing at 4 ℃ for 30 minutes to promote delamination. Then, 13000g of the supernatant is centrifuged for 15 minutes at 4 ℃, all the upper layer lipid extract is quantitatively transferred to a 96-well plate, and the 96-well plate is placed in a nitrogen blowing instrument for drying; and after the sample is dried, adding 200 microliters of isopropanol-acetonitrile (1/1, v/v) solvent, uniformly mixing for 1 minute in a vortex manner, carrying out sample redissolution, carrying out centrifugal treatment on the sample for 5 minutes at 4 ℃ under 2200g conditions after the redissolution is finished, respectively transferring 50 microliters of supernatant into a PCR plate after the centrifugation is finished, and waiting for mass spectrometry detection and analysis.

In some embodiments, the second control group extraction scheme is set to: n-butanol-methanol (1: 1) extraction system.

The detection process comprises the following steps: placing 100 microliters of the plasma sample in a 0.5ml 96-well plate, adding a 3-fold volume of n-butanol-methanol (1: 1, v/v) solvent, uniformly mixing for 5 minutes by vortex, and extracting lipid molecules; after complete extraction, the sample is centrifuged for 5 minutes at 4 ℃ and 2200 g; after the centrifugation is finished, taking supernatant in the sample, transferring the supernatant into a protein precipitation plate, and then centrifuging the protein precipitation plate for 5 minutes at 4 ℃ under 2200 g; placing the filtrate sample collected after the centrifugation in a nitrogen blowing instrument for blow-drying; and after the sample is dried, adding 100 microliters of isopropanol-acetonitrile (1/1, v/v) solvent, uniformly mixing for 1 minute in a vortex manner, carrying out sample redissolution, carrying out centrifugal treatment on the sample for 5 minutes at 4 ℃ under 2200g conditions after the redissolution is finished, respectively transferring 50 microliters of supernatant into a PCR plate after the centrifugation is finished, and waiting for mass spectrometry detection and analysis.

In some embodiments, the extraction system of the third control group is set as: n-butanol-methanol (3: 1) extraction system.

The detection process comprises the following steps: placing 100 microliters of the plasma sample in a 0.5ml 96-well plate, adding a 3-fold volume of n-butanol-methanol (3: 1, v/v) solvent, uniformly mixing for 5 minutes by vortex, and extracting lipid molecules; after the extraction is completed, the sample is centrifuged for 5 minutes at 4 ℃ and 2200 g; after the centrifugation is finished, taking supernatant in the sample, transferring the supernatant into a protein precipitation plate, and then centrifuging the protein precipitation plate for 5 minutes at 4 ℃ under 2200 g; placing the filtrate sample collected after the centrifugation in a nitrogen blowing instrument for blow-drying; and after the sample is dried, adding 100 microliters of isopropanol-acetonitrile (1/1, v/v) solvent, uniformly mixing for 1 minute in a vortex manner, carrying out sample redissolution, carrying out centrifugal treatment on the sample for 5 minutes at 4 ℃ under 2200g conditions after the redissolution is finished, respectively transferring 50 microliters of supernatant into a PCR plate after the centrifugation is finished, and waiting for mass spectrometry detection and analysis.

In some embodiments, the extraction system for the experimental group is set as: an isopropanol extraction system.

The detection process comprises the following steps: placing 100 microliters of the plasma sample in a 0.5ml 96-well plate, adding 3 times volume of isopropanol solvent, uniformly mixing for 5 minutes in a vortex manner, and extracting lipid molecules; after the extraction is completed, the sample is centrifuged for 5 minutes at 4 ℃ and 2200 g; after the centrifugation is finished, taking supernatant in the sample, transferring the supernatant into a protein precipitation plate, and then centrifuging the protein precipitation plate for 5 minutes at 4 ℃ under the condition of 2200 g; after centrifugation, 50. mu.l of each supernatant was transferred to a PCR plate and analyzed by mass spectrometry.

In some embodiments, the detection conditions under which the lipidomic sample is subjected to mass spectrometric detection analysis are set as:

the sample detection adopts an Ultimate 3000 ultra-high performance liquid chromatograph and a Q active square-Orbitrap high-resolution mass spectrometer system (Thermo Scientific, USA); lipid sample separation an Acquity BEH C18 chromatography column (Waters, 2.1X 100mm,1.7 μm) was used, wherein the column temperature was set at 50 ℃; mobile phase A contained 2mM ammonium formate, ACN: H₂O60: 40, 0.1% FA, mobile phase B contained 2mM ammonium formate, ACN IPA 10:90, 0.1% FA, and gradient elution conditions are shown in table 1 below.

The structure identification of the lipid metabolite is carried out by DDA secondary mass spectrum, and the detection parameters of the ionized lipid molecule mass spectrum are as follows: the mass spectrum full scan resolution is 70,000@ m/z 200, the AGC is 1E6, the maximum ion injection time is 100ms, the scanning range is m/z 200-2000, the standardized collision energy is 27%, the secondary mass spectrum scanning resolution is 17,500@ m/z 200, the scanning range is m/z 200-2000, the AGC is 1E5, the maximum ion injection time is 50ms, and the dynamic exclusion time is 4 s; and the optimized positive ion detection modes all adopt 20% + 30% step normalization collision energy.

Table 1 gradient elution table:

figure 2 shows mass spectrum results for the control and experimental groups shown according to the exemplary embodiment, as shown in figure 2, the MTBE-methanol-water extraction system showed the least peaks over the 10-18 minute gradient compared to the other three systems, whereas the isopropanol extraction system showed more peaks and also higher response intensity over this time period.

Fig. 3 shows a statistical analysis chart of the number of lipid molecules and coverage types according to an exemplary embodiment, as shown in fig. 3, a spectrogram obtained by detecting different systems is subjected to library search processing by using Lipidsearch software of Thermo Fisher corporation, and experimental statistics results are as follows, compared with other three extraction systems, the isopropanol extraction system obtains the largest number of lipid molecules (737 types) and more identified lipid types (20 types) after library search identification. By combining the two results, the isopropanol extraction system can be obtained, compared with other systems, the safety is improved (toxic reagents such as MTBE, chloroform and the like are avoided), the experimental operability is simplified (objects to be detected do not need to be absorbed by layering), meanwhile, the pretreatment time is saved (extraction solvent does not need to be prepared), and the isopropanol can be compatible with the mobile phase of a mass spectrometer, so that the link of sample redissolution can be saved, and the lipid extraction efficiency and the lipid species coverage are improved.

The present application also shows a lipidomic sample pretreatment device, which is suitable for use in the methods of the above embodiments.

In some embodiments, the device is an automated workstation. Fig. 4 shows a software schematic of an automation workstation. Fig. 5 shows an internal block diagram of the automation workstation. As shown in fig. 5, the internal structure of the automated workstation includes: 1-constant temperature mixing shaking table, 2-200 microliter gun head, 3-PCR plate, 4-50 microliter gun head, 5-0.5mL 96-well plate, 7-12 channels of groove, and 9-protein precipitation plate.

In some embodiments, taking 480 clinical large-queue samples processed by an automated workstation as an example, in combination with an automated sample pretreatment system proposed in this project, a sample lipidomic pretreatment process is specifically integrated into the automated workstation, and the whole automated process can be divided into four parts: sample subpackaging, target object extraction, protein filtration, filtrate subpackaging and transferring.

The automatic processing system comprises a sample storage module, a processing solution supply module, a protein precipitation plate supply module, a sample suction module, a pretreatment reaction container (96 pore plate) supply module, a shaking table module, a centrifugation module and a PCR plate supply module, wherein the sample storage module, the processing solution supply module, the protein precipitation plate supply module, the sample suction module, the pretreatment reaction container (96 pore plate) supply module, the shaking table module, the centrifugation module and the PCR plate supply module are all electrically connected with a control terminal to realize automatic control.

The processing liquid supply unit includes an extraction solvent supply module and an internal standard supply module. The processing liquid supply unit may be 4-channel, 8-channel, or 12-channel, and each channel is provided with a different reagent supply. In this embodiment, the control terminal is a computer. The functions of automatic sample split charging, sample loading, vibration, centrifugation and the like are realized through the control terminal. Specifically, the automated processing system performs the automated processing flow of the sample as follows:

uniformly mixing an experiment sample added with an isopropanol solvent in a vortex manner under a first condition;

In the specific implementation, taking an experimental sample as a plasma sample as an example, an automatic workstation is used for automatically transferring 100 microliters of the plasma sample to a 0.5 milliliter 96-well plate and placing the plate on a 1-position constant-temperature mixing shaker, absorbing 3 times of volume of isopropanol solution of the plasma sample from a 7-position 12-channel first row, adding the isopropanol solution into the 1-position plasma sample, and performing vortex mixing at the rotating speed of 1000rpm for 5 minutes to extract lipid molecules.

And centrifuging the test sample after vortex mixing at low temperature under a second condition to obtain the supernatant of the micro sample.

In a specific implementation, a 0.5ml 96 well plate on a constant temperature mixing shaker at position 1, in which a plasma sample is placed, is centrifuged at 4 ℃ and 2200g for 5 minutes.

The supernatant was transferred to a protein precipitation plate.

In specific implementation, after the centrifugation is finished, the 0.5ml 96-well plate is placed back on the 1-position constant-temperature mixing shaking table, and then the supernatant obtained by centrifuging the plasma sample in the 0.5ml 96-well plate is transferred to the 9-position protein precipitation plate.

The supernatant in the protein precipitation plate was centrifuged at low temperature under a third condition to obtain a filtrate.

The filtrate was determined as a lipidomics sample.

In the specific implementation, the filtrate is placed on a constant-temperature mixing shaking table at the No. 1 position, 50 microliters of the filtrate is transferred and placed on a PCR plate at the No. 3 position, and the lipidomics sample is determined to wait for mass spectrometry detection and analysis.

The more detailed pretreatment experimental procedure using the automated workstation is shown in table 2:

table 2 automated process flow:

the mass spectrometric detection parameters were as follows:

in some embodiments, the experimental sample detection is performed using an Ultimate 3000 ultra-high performance liquid chromatograph with a Q active square-Orbitrap high resolution mass spectrometer system (Thermo Scientific, USA); lipid sample separation an Acquity BEH C18 chromatography column (Waters, 2.1X 100mm,1.7 μm) was used, wherein the column temperature was set at 50 ℃; mobile phase a was 2mM ammonium formate, ACN: H2O: 60:40, 0.1% FA, mobile phase B was 2mM ammonium formate, ACN: IPA: 10:90, 0.1% FA, and the gradient elution conditions were as shown in table 1 above.

Carrying out structure identification on the lipid metabolite by DDA secondary mass spectrum, and detecting parameters of ionized lipid molecule mass spectrum: the mass spectrum full scan resolution is 70,000@ m/z 200, the AGC is 1E6, the maximum ion injection time is 100ms, the scanning range is m/z 200-2000, the standardized collision energy is 27%, the secondary mass spectrum scanning resolution is 17,500@ m/z 200, the scanning range is m/z 200-2000, the AGC is 1E5, the maximum ion injection time is 50ms, and the dynamic exclusion time is 4 s; and the optimized positive ion detection modes all adopt 20% + 30% step normalization collision energy.

It should be noted that the automatic workstation can be equipped with 4, 8, 12 or 96 channels to realize microliter-level accurate pipetting, and automatically perform pretreatment experiments according to a set program, so that experimental errors caused by artificial fatigue can not occur; meanwhile, the workstation repeatedly carries out a plurality of experiments, the result uniformity and the reproducibility degree are very high, and the automation equipment can work unattended, so that the efficiency of the pretreatment experiment is improved to a certain extent.

Fig. 6 shows a lipid intensity distribution ranking chart of all samples in an exemplary embodiment, as shown in fig. 6, after the detection of the sample is completed, firstly, the quantitative intensity (intensity) of all lipid molecules in the sample is subjected to statistical analysis, and the result shows that the quantitative intensity of all identifiable lipid molecules in the sample spans 4 orders of magnitude, the intensity (Log 10) can detect the intensity of fragments from lower 6 to higher 10, and the intensity distribution is stable, which indicates that the data coverage depth is wide, and the detection can be covered from low-content lipid molecules to high-content lipid molecules, so that the pre-treatment method can be used for the later analysis.

Fig. 7 is a statistical graph showing the identification number of the sample lipid molecules according to the exemplary embodiment, and as shown in fig. 7, the majority of the lipid molecules identified by the sample are distributed around 750, and the identification number is relatively stable, indicating that the reproducibility and stability of the pretreatment method are high.

Fig. 8 shows a statistical chart of the total intensity of lipid molecules obtained by lipidomics identification of a part of samples according to the exemplary embodiment, and as shown in fig. 8, the distribution of the total intensity of lipid identification obtained by 120 samples is very stable and has small fluctuation range, which indicates that the stability of the whole pretreatment result can be obviously improved by utilizing an automatic pretreatment workstation in a lipidomic pretreatment flow.

Finally, it should be noted that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.

The foregoing description, for purposes of explanation, has been presented in conjunction with specific embodiments. However, the illustrative discussions above are not intended to be exhaustive or to limit the embodiments to the precise forms disclosed above. Many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles and the practical application, to thereby enable others skilled in the art to best utilize the embodiments and various embodiments with various modifications as are suited to the particular use contemplated.

Claims

1. A method for pre-processing a lipidomic sample, said method comprising:

centrifuging the test sample after vortex mixing at low temperature under a second condition to obtain supernatant of the micro sample;

transferring the supernatant to a protein precipitation plate;

centrifuging the supernatant in the protein precipitation plate at a low temperature under a third condition to obtain a filtrate;

the filtrate was determined as a lipidomics sample.

2. The lipidomic sample pretreatment method of claim 1, wherein the vortexing and mixing the isopropanol solvent-added experimental sample under a first condition comprises:

the volume ratio of the experimental sample to the isopropanol solvent is 1: 3 to 1: 4.

3. the lipidomic sample pretreatment method of claim 1, wherein said first condition is configured to homogenize the test sample at 1000-2200 rpm for 5-10 minutes.

4. The lipidomic sample pretreatment method of claim 1, wherein said second condition is configured to centrifuge said test sample at 4-8 ℃ for 5-10 minutes at 1000-2200 g.

5. The lipidomic sample pretreatment method of claim 1, wherein said third condition is configured to centrifuge said test sample at 4-8 ℃ for 5-10 minutes at 1000-2200 g.

6. A method for detecting a lipidomic sample, said method comprising:

obtaining a lipidomic sample, wherein the lipidomic sample is subjected to sample pretreatment according to the method of any one of claims 1 to 5;

and carrying out mass spectrum detection analysis on the lipidomic sample to obtain a detection result of the lipidomic sample.

7. A lipidomic sample pretreatment device, characterized in that said device is suitable for use in the method according to any of claims 1 to 5.

8. The lipidomic pre-sample processing apparatus of claim 7, wherein the apparatus is an automated workstation.