Spatial resolution lipidomics method based on zebra fish

Abstract

The invention discloses a zebra fish-based spatial resolution lipidomics method, and belongs to the technical field of biological medicines. The zebrafish-based spatial resolution lipidomics method provided by the invention comprises the following steps: separating a zebrafish trunk from yolk; sample pretreatment and chromatography-mass spectrometry detection. According to the method, yolk is sucked by a pipettor, trunks are cleaned in batches by a vortex instrument, after the trunks and the yolk are pretreated, a sample is subjected to lipidomics detection by an ultra-high performance liquid chromatography tandem quadrupole electrostatic field orbitrap high-resolution mass spectrometer, and further data analysis is performed. According to the method, the trunk and yolk samples which can be used for high-quality and high-coverage lipidomics analysis are obtained while high sample flux is maintained for the first time, and regionalized analysis of zebrafish embryo / larva lipid metabolism is realized. The method has the advantages of simple sample mechanism, low lipid signal interference, high sensitivity and the like, and can detect inrecognizable subtle changes and key lipid molecules in the whole sample.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a spatial resolution lipidomics method based on zebrafish. BACKGROUND

[0002] Lipidomics is an important branch of metabolomics, aiming to comprehensively identify and quantitatively analyze all lipid molecules in the body, and to deeply study their biological functions, interactions and dynamic change rules. In recent years, lipid metabolism research has gradually developed from static characterization to dynamic analysis. Researchers are more deeply exploring the complex regulation mechanism of lipid metabolism in physiological and pathological processes, and actively promoting the transformation of these basic findings into new disease markers, treatment strategies and drug research and development.

[0003] Zebrafish, as a typical model organism for lipid metabolism research, has transparent embryos and rapid development, allowing researchers to directly, real-time and non-invasively observe lipid storage, transport and metabolism. In addition, the advantages of large embryo yield and short development cycle make large-scale pharmacological screening, environmental toxicant evaluation and functional lipid effect research feasible and cost-effective. More importantly, zebrafish eggs rely entirely on endogenous yolk nutrition during the early life stage, without the need for exogenous food intake, effectively eliminating the interference of factors such as feeding behavior, digestion and absorption, and microbiota, providing a unique and pure model system for studying endogenous lipid mobilization, lipid metabolism programming and its regulation mechanism during embryonic and early development stages. However, zebrafish embryos have a significant zonal structure, and there are significant differences in lipid composition, function and dynamic changes between the body and yolk. Mixing analysis of the two may mask a lot of important biological information. To further study the regulation mechanism of lipid metabolism, the function of lipids in development, and the model of lipid-related diseases, it is necessary and more optimal to separate the zebrafish embryo or larva body (Body) and yolk (Yolk) for lipid identification. At present, the separation method for zebrafish embryo or larva body (Body) and yolk (Yolk) is difficult to balance between throughput and quality, and the lipid identification components are single and limited in coverage.

[0004] Therefore, it is urgent to develop a spatial resolution lipidomics method based on zebrafish, which has great significance for omics research. SUMMARY

[0005] The present application discloses a spatial resolution lipidomics method based on zebrafish, aiming to solve the technical problem that the existing separation method for zebrafish embryo or larva body (Body) and yolk (Yolk) is difficult to balance between throughput and quality, and the lipid identification components are single.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is: The first aspect of the present application provides a zebrafish-based spatially resolved lipidomics method, the method comprising: The zebrafish washed with PBS buffer is placed on agarose gel, and the physical demembranation is performed under a body microscope to obtain demembranated zebrafish; The body position of the demembranated zebrafish is adjusted so that the tail is downward, the yolk sac is rightward, the zebrafish is laterally lying, and the yolk is regularly arranged to obtain twice-treated zebrafish; The twice-treated zebrafish is collected, vortexed in PBS buffer, and the trunk is cleaned after PBS buffer cleaning to obtain the trunk and yolk, which are collected and stored in liquid nitrogen, respectively. The trunk and yolk are pretreated, and after lipidomics detection of the samples by ultra-high performance liquid chromatography tandem quadrupole electrostatic field orbitrap high-resolution mass spectrometer, data analysis is performed.

[0007] In combination with the first aspect, preferably, the zebrafish comprises 1-23 hpf zebrafish embryos, 24-72 hpf zebrafish embryos, and zebrafish larvae of more than 72 hpf.

[0008] In combination with the first aspect, preferably, if the zebrafish is a zebrafish larvae of more than 72 hpf, the zebrafish washed with PBS buffer is placed on agarose gel, and after being placed under a body microscope, the physical demembranation step does not need to be performed.

[0009] In combination with the first aspect, preferably, if the zebrafish is a 1-23 hpf zebrafish embryo, the germ layer cells of the demembranated zebrafish are separated from the yolk to obtain the trunk and yolk, which are collected and stored in liquid nitrogen, respectively.

[0010] In combination with the first aspect, preferably, the pretreatment of the trunk and yolk comprises: The obtained trunk and yolk are added into an extractant for extraction, centrifuged, vacuum concentrated, added into a redissolving solution for redissolution, and after centrifugation, the pretreatment is completed.

[0011] In combination with the first aspect, preferably, the extractant is a mixed solution of chloroform and methanol with a volume ratio of 2:1, and 0.01% lipidomics analysis internal standard; The redissolving solution is a mixed solution of isopropyl alcohol, acetonitrile, and water with a volume ratio of isopropyl alcohol: acetonitrile: water = 6:13:1.

[0012] In combination with the first aspect, preferably, after the pretreatment of the trunk and yolk, the lipidomics detection of the samples by ultra-high performance liquid chromatography tandem quadrupole electrostatic field orbitrap high-resolution mass spectrometer comprises: The liquid phase conditions were as follows: Mobile phase A: a mixed solution of 40% water and 60% acetonitrile; Mobile phase B: a mixed solution of 90% isopropanol and 10% acetonitrile; In positive ion mode, 0.1% formic acid and 10 mmol·L⁻¹ were added to both mobile phases. -1 Ammonium formate, in negative ion mode, 10 mmol·L⁻¹ was added to both mobile phases. -1 Ammonium acetate.

[0013] In conjunction with the first aspect, preferably, when the torso and yolk are pretreated, and then lipidomics detection is performed on the samples using ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbital trap high-resolution mass spectrometry, the following steps are included: Gradient elution: 0 min, 37% B; 0–1.5 min, 37% B; 1.5–15.6 min, B linearly increases to 85%; 15.6–18 min, 97% B; 18–18.2 min, returns to the initial mobile phase ratio of 37% B; 18.2–20 min, 37% B.

[0014] In conjunction with the first aspect, preferably, when the torso and yolk are pretreated, and then lipidomics detection is performed on the samples using ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbital trap high-resolution mass spectrometry, the following steps are included: An electrospray ionization source was used, scanning in both positive and negative ion modes; the scanning range was m / z 200–1700, with a spray voltage of 3.5 kV for positive ion mode and 2.8 kV for negative ion mode; the sheath gas pressure was 35 AU; the auxiliary gas pressure was 15 AU; the backup gas pressure was 0 AU; the capillary temperature was 300 ℃; the ion transmission tube temperature was 320 ℃; the scanning mode was full scan, with a resolution set to 120,000 full width at half maximum (FWHM); and data-dependent mass spectrometry (ddMS) was employed. 2 ), and fragment detection experiments were conducted on the parent ion.

[0015] In conjunction with the first aspect, preferably, the data analysis includes: The test data were compared and analyzed with MS / MS fragment maps, mzVault, and MassList database information to identify lipid metabolites and determine the number and types of lipids.

[0016] Compared with the prior art, the advantages or beneficial effects of the embodiments of this application include at least the following: The analytical method provided in this application obtains lipidomics data by efficiently separating the zebrafish trunk and yolk sac. On the one hand, it can efficiently separate the zebrafish trunk and yolk sac and is applicable to zebrafish at different developmental stages; while ensuring high sample throughput, it can obtain trunk and yolk sac samples that meet the requirements of high-quality, high-coverage lipidomics analysis, thereby achieving "regional" analysis of zebrafish lipid metabolism. On the other hand, detecting lipidomics information in zebrafish embryos / larvae has a simple sample mechanism, low lipid signal interference, and reduces the lipid complexity of individual samples. After separation, the lipid profile of the trunk's own synthesis and metabolism can be directly analyzed, avoiding interference from lipids stored in the yolk sac, and detecting subtle changes and key lipid molecules that cannot be identified in the whole sample. Simultaneously, the analytical method used in this application can be combined with lipidomics data to precisely locate the source of lipid metabolism abnormalities, providing precise spatial clues for revealing molecular mechanisms. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This image shows the effect of separating the zebrafish embryo / larvae's body from the yolk sac in an embodiment of this application. Figure 2 A comparative diagram of lipid numbers and categories in 5.25 hpf-120 hpf zebrafish embryos / larvae Body & Yolk and Embryo, provided for embodiments of this application; Figure 3 Principal component analysis plots and cluster heatmaps of Body, Yolk, and Embryo at different developmental stages prepared for embodiments of this application; Figure 4 Volcano plots showing differential lipid profiles of Body, Yolk, and Embryo at different developmental stages prepared for embodiments of this application; Figure 5 The diagram shows the dynamic changes in lipid content in the Body, Yolk, and Embryo of 5.25 hpf-120 hpf zebrafish embryos / larvae, provided for embodiments of this application. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0020] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0021] In the following description of this embodiment, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0022] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0023] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0024] It should be noted that all raw materials and / or reagents in the embodiments of this application were purchased on the market or prepared according to conventional methods known to those skilled in the art.

[0025] In a first aspect, embodiments of this application provide a spatially resolved lipidomics method based on zebrafish, the method comprising: Zebrafish washed with PBS buffer were placed on agarose gel and physically demetabolized under a stereomicroscope to obtain demetabolized zebrafish. Adjust the body position of the zebrafish after molting so that its tail is facing down, its yolk sac is facing right, it is lying on its side, and its yolk sac is arranged regularly. Extract the yolk sac to obtain a secondary-processed zebrafish. Collect the zebrafish that have undergone secondary treatment, add them to PBS buffer and vortex them, wash the cleaned body with PBS buffer to obtain the body and yolk, collect them separately and store them in liquid nitrogen; After pretreatment of the torso and egg yolk, the samples were subjected to lipidomics detection and data analysis using ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbital trap high-resolution mass spectrometry.

[0026] In this embodiment, the zebrafish is preferably zebrafish embryos at 1-23 hpf, zebrafish embryos at 24-72 hpf, or zebrafish larvae at 72 hpf or more, and more preferably embryos or larvae at different growth stages such as 5.25 hpf, 10 hpf, 24 hpf, 48 hpf, 72 hpf, 96 hpf, and 120 hpf. This application establishes a microscopic separation method for the trunk and yolk sac of zebrafish embryos / larvae at different developmental stages, enabling "regional" analysis of lipid metabolism in zebrafish embryos / larvae.

[0027] In this embodiment, if the zebrafish are zebrafish larvae with a growth rate of 72 hpf or higher, after washing with PBS buffer, the zebrafish are placed on an agarose gel and examined under a stereomicroscope; no physical demetabolism step is required. That is, zebrafish larvae that have already hatched do not require a demetabolism step.

[0028] In this embodiment, if the zebrafish is a 1-23 hpf zebrafish embryo, the germ cells of the demembranous zebrafish are torn apart from the yolk to obtain the trunk and yolk, which are collected separately and stored in liquid nitrogen. This eliminates the need for vortexing in PBS buffer.

[0029] It should be noted that the zebrafish embryo / larval separation method provided in this application employs a pipette to aspirate yolk in batches and a vortex mixer to clean the body in batches. This method achieves, for the first time, high-throughput sample collection of body and yolk samples suitable for high-quality, high-coverage lipidomics analysis, realizing "regional" analysis of lipid metabolism in zebrafish embryos / larvae. Furthermore, the spatially resolved lipidomics method based on zebrafish embryos provided in this application systematically establishes and optimizes standardized pretreatment and mass spectrometry detection procedures applicable to samples from different anatomical sites such as whole embryos, body, and yolk. This enables high-coverage identification and precise quantification of lipid composition in various regions of the zebrafish embryo in a spatial dimension, thereby supporting comparative studies of differences in lipid metabolism between different tissue regions.

[0030] It should be noted that: 1. This application establishes a microscopic separation method for the trunk and yolk of zebrafish embryos / larvae at different developmental stages. While maintaining high sample throughput, it obtains trunk and yolk samples suitable for high-quality, high-coverage lipidomics analysis, achieving "regional" analysis of lipid metabolism in zebrafish embryos / larvae. This is of great significance for in-depth research on the regulatory mechanisms of lipid metabolism and the function of lipids in development. 2. This application has the advantages of simple sample mechanism and low lipid signal interference in detecting lipidomics information in zebrafish embryos / larvae, reducing the lipid complexity of individual samples. After separation, the lipid profile of synthesis and metabolism in the trunk itself can be directly analyzed, avoiding interference from lipids stored in the yolk. It can detect subtle changes and key lipid molecules that cannot be identified in the whole sample. 3. This application improves the accuracy and specificity of biomarker discovery. Lipidomics analysis after separating the trunk and yolk of zebrafish embryos / larvae can accurately locate the source of lipid metabolism abnormalities. If the changes are mainly in the yolk, it suggests impaired lipid absorption, storage, or mobilization; if in the trunk, it indicates defects in transport, uptake, or intracellular metabolism, providing precise spatial clues for revealing the molecular mechanism of action. 4. This application facilitates more precise absolute quantification of lipids. Separating the zebrafish embryo / larval trunk from the yolk and quantifying them separately allows for the acquisition of the true content and distribution differences of lipids in each region, which is crucial for constructing metabolic models and understanding lipid function. 5. This method significantly improves the experimental efficiency of zebrafish in environmental toxicity assessment and large-scale pharmacological screening. Its core lies in the use of efficient batch processing technology: batch yolk is aspirated using a pipette, and the zebrafish trunk is cleaned in batches using a vortex mixer. In practice, a single vortex treatment (30-60 seconds) can complete the cleaning of residual yolk from the trunks of 50-100 zebrafish. Compared with existing processing methods, this method not only significantly improves operational efficiency but also easily obtains a larger number of experimental samples.

[0031] The technical solution of this application will be further described below with reference to specific embodiments.

[0032] Example 1 This embodiment provides zebrafish embryos at different developmental stages.

[0033] S101: Zebrafish Embryo Acquisition: Zebrafish embryos were obtained through artificial insemination. The collected fertilized embryos were washed with embryo culture medium to remove attached impurities and microorganisms. After washing, the fertilized embryos were examined microscopically, and abnormally developed or damaged embryos were discarded to ensure the homogeneity of subsequent experimental materials. Important developmental time points in zebrafish embryonic development (5.25 hpf, 10 hpf, 24 hpf, 48 hpf, 72 hpf, 96 hpf, 120 hpf) were selected, with five parallel samples per group. Sixteen Body, Yolk, or Embryo samples were collected from each parallel sample for lipidomics analysis. Zebrafish Body, Yolk, and Embryo samples at different developmental stages were collected according to the following procedures.

[0034] S102: Zebrafish embryo sample isolation (1) Technique for separating the trunk and yolk of zebrafish embryos at 5.25 hpf and 10 hpf; (a) Select zebrafish embryos that are at a suitable developmental stage and are normally fertilized, and wash them with PBS buffer to remove impurities; (b) Transfer the zebrafish embryos to be separated to a 60 mm culture dish covered with agarose (with an appropriate amount of PBS solution in the culture dish), and place the other embryos to be separated on ice to delay development. (c) Move the culture dish to the center of the stereomicroscope field of view and adjust the embryos so that they are regularly arranged in the center of the field of view; (d) Use vascular forceps to physically demembranes the embryo. After demembranes are demembranes, gently grasp the germ cells of the embryo with vascular forceps and tear the germ cells from the yolk. Control the tearing force to ensure that the yolk is not punctured and keep the germ cells as intact as possible. (e) Collect the separated zebrafish embryo trunk and yolk using a 20 μL pipette, flash freeze in liquid nitrogen, and store at -80 °C for subsequent experiments.

[0035] (2) Technique for separating the yolk sac from the body of 24 hpf embryos to 96 hpf larvae (a) Select healthy embryos / larvae at the appropriate developmental stage. Examine the morphological characteristics of the embryos / larvae under a microscope to ensure that there are no abnormalities in their development. Wash with PBS buffer to remove impurities. Place the embryos in an ice bath for 15 minutes to reduce their activity and facilitate subsequent operations. (b) After the ice bath, place the embryos / larvae in a 60 mm culture dish covered with agarose (with an appropriate amount of PBS solution in the dish). Move the culture dish to the center of the stereomicroscope field of view and adjust the embryos / larvae to be regularly arranged in the center of the microscope field of view. Use vascular forceps and a separation needle to physically detach the 24 hpf and 48 hpf zebrafish embryos; the 72 hpf and 96 hpf zebrafish larvae that have already hatched do not need to undergo the detachment step. (c) Use a 20 μL pipette to aspirate the complete yolk sac, adjust the body position of the zebrafish embryo / larva after demembranous detachment so that its tail is facing down and the yolk sac is facing right, lay it on its side, press down the pipette, align the pipette tip with the embryo / larva yolk sac, ensure that the yolk sac is completely embedded in the pipette tip, quickly release the pipette to aspirate and separate the yolk sac completely; (d) Use a vortex apparatus to remove residual yolk from the trunks of zebrafish larvae at 24 hpf, 48 hpf, 72 hpf, or 96 hpf. Transfer the zebrafish larvae trunks into 1.5 mL low-adsorption EP (Eppendorf Tube, EP) tubes, add 1 mL of PBS to the EP tubes, vortex for 30 s, wash with PBS buffer, and examine under a microscope to ensure no yolk remains on the larvae trunks. Collect the separated zebrafish embryo trunks and yolk using a 20 μL pipette, flash freeze in liquid nitrogen, and store at -80 ℃ for subsequent experiments.

[0036] (3) Technique for separating the trunk and yolk sac of 120 hpf larvae (a) Select healthy larvae at the appropriate developmental stage, examine the morphological characteristics of the larvae under a microscope to ensure that there are no abnormalities in the development of the larvae, wash them with PBS buffer to remove impurities, and put the larvae in an ice bath for 15 minutes to reduce the activity of the larvae and facilitate subsequent operations. (b) After the ice bath, the larvae were placed in a 60 mm culture dish covered with agarose (with an appropriate amount of PBS solution in the culture dish). The culture dish was moved to the center of the field of view of the stereomicroscope, and the larvae were adjusted to be arranged regularly in the center of the field of view of the microscope. Another part of the larvae waiting to be separated were given an ice bath to slow down cell metabolic activity and inhibit development. (c) Insert one side of the vascular forceps into the connection between the yolk and the body, and quickly slice along the connection towards the tail of the yolk to separate the yolk from the body of the zebrafish larva. Remove the vascular forceps and use the tip of the vascular forceps to cut off the connection between the yolk and the body. (d) Use a vortex apparatus to clean the residual yolk sac on the body of 120 hpf zebrafish larvae. Transfer the larval body to a 1.5 mL low-adsorption EP tube, add 1 mL PBS to the EP tube, vortex for 30 s, wash with PBS buffer, and under microscopic examination, no yolk sac is found on the larval body. Collect the body and yolk sac separately, flash freeze in liquid nitrogen, and store at -80 ℃ for subsequent experiments. S103: Sample Pretreatment (1) Add 200 μL of a mixture of chloroform and methanol containing 0.01% lipidomics analysis internal standard to each sample tube, with a volume ratio of 2:1. Add three 1 mm diameter homogenization beads, homogenize in a tissue homogenizer at 70 Hz for 2 min, vortex at low temperature for 5 min, and sonicate in an ice bath for 3 min. The entire extraction process should not exceed 10 ℃.

[0037] (2) After sonication, add 40 μL of pre-cooled ultrapure water to each sample tube, vortex at low temperature for 30 s, centrifuge at 12500 rpm and 4 ℃ for 5 min to promote separation, take 100 μL of the clear liquid into a low adsorption EP tube, and concentrate under vacuum for 7 min. (3) After reconstitution with 100 μL of reconstitution solution (isopropanol: acetonitrile: water = 6: 13: 1, v / v / v), vortex at 2000 rpm for 5 min, transfer all the liquid to the inner tube, centrifuge at 12500 rpm and 4 ℃ for 5 min, take 70 μL of supernatant into a new inner tube, and store it in a -20 ℃ refrigerator for later testing; S104: LC-MS / MS detection After sample pretreatment, lipidomics analysis was performed on the samples using an ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbital trap high-resolution mass spectrometer (UHPLC-Orbitrap Exploris 240). Quality control (QC) samples were obtained by mixing equal volumes of each sample to confirm the reproducibility of the lipidomics analysis.

[0038] Liquid chromatography conditions: Hypersil Gold C18 column (2.1 mm × 100 mm, 1.7 μm); column temperature 40 ℃; injection volume 5 μL; sample chamber temperature 10 ℃. Mobile phase A: a mixture of 40% water and 60% acetonitrile; mobile phase B: a mixture of 90% isopropanol and 10% acetonitrile; in positive ion mode, 0.1% formic acid and 10 mmol·L⁻¹ were added to both mobile phases. -1 Ammonium formate, in negative ion mode, 10 mmol·L⁻¹ was added to both mobile phases. -1 Ammonium acetate.

[0039] Gradient elution program: 0 min, 37% B; 0–1.5 min, 37% B; 1.5–15.6 min, B linearly increases to 85%; 15.6–18 min, 97% B; 18–18.2 min, return to initial mobile phase ratio (37% B); 18.2–20 min, 37% B.

[0040] Mass spectrometry conditions: Electrospray ionization (ESI) source, scanning in both positive and negative ion modes; scan range m / z 200–1700, spray voltage 3.5 kV (positive ion mode) and 2.8 kV (negative ion mode); sheath gas pressure 35 AU; auxiliary gas (N2) pressure 15 AU; backup gas (N2) pressure 0 AU; capillary temperature 300 ℃; ion transmission tube temperature 320 ℃; full scan mode (Full MS), resolution set to 120,000 full width at half maximum (FWHM); data-dependent secondary mass spectrometry (ddMS) mode. 2 The top four precursor ions with the highest response intensity in the full scan were selected for fragmentation analysis, and the lower limit of the precursor ion response for triggering fragmentation detection was set to 5 × 10⁻⁶. 3 The dynamic exclusion time is 5 seconds. MS 2 Scanning was performed at a resolution of 30,000 FWHM, with a parent ion isolation width of 1.5 m / z and a normalized collision mode (HCD collision energy of 27.5%).

[0041] Preliminary data processing: Ion information acquired by LC-MS was imported into Compound Discover 3.3 software. Based on the retention time (RT) of the lipidomics analysis internal standard, MS / MS fragment maps, and information from the mzVault and MassList databases, lipid metabolites were preliminarily screened.

[0042] Using the lipidomics method described in this application, key developmental time points from 5.25 hpf in zebrafish embryos to 120 hpf in zebrafish larvae were selected for lipidome analysis. The resulting images of the separated lipids are shown below. Figure 1 As shown, the results of the lipidomics assay are as follows: Figure 2 As shown, the Body and Yolk lipid assays detected 2156 lipids and 59 lipid types, while the Embryo lipid assay detected 1561 lipids and 56 lipid types. This demonstrates that this method detects a greater number and variety of lipids, exhibits higher sensitivity, and can accurately pinpoint the source of lipid abnormalities.

[0043] according to Figure 3It was found that zebrafish at different developmental stages exhibited different lipidomical characteristics. Principal component analysis (PCA) plots showed a clear distinction between the lipidomical characteristics of the 24 hpf body and those of the 5.25 hpf and 10 hpf bodies, suggesting that the lipid composition of the zebrafish embryo's body undergoes significant changes after reaching this stage of development. However, in the embryonic body, the zebrafish body is influenced or covered by lipids from the yolk, thus obscuring this significant change that occurred in the 24 hpf body. Clustering heatmap results showed that the technical reproducibility of zebrafish samples from different developmental stages was tightly clustered, indicating that the sampling method used in this application has excellent reproducibility and stability.

[0044] according to Figure 4 Analysis of lipid dynamics during zebrafish embryonic development revealed that lipid profile changes in the embryo do not accurately reflect the independent dynamics of the body and yolk regions. Specifically, before 72 hpf, lipids in the body continuously accumulated and then stabilized until 96 hpf, subsequently declining; while lipids in the yolk were continuously consumed. These independent and distinct patterns of change suggest that measurements at the whole embryonic level mask the true lipid dynamics of different tissue compartments.

[0045] according to Figure 5 It is known that the lipid content in different compartments (Body and Yolk) of zebrafish embryos exhibits distinctly different dynamic patterns during development (5.25 hpf-120 hpf). However, the lipid content curve of Embryo mainly reflects the overall effect of net lipid transfer from Yolk to Body, rather than the independent changes in Body and Yolk.

[0046] To investigate the effects of perfluorooctane sulfonate (PFOS) exposure on lipid metabolism in zebrafish embryos using spatially resolved lipidomics, tests were conducted, and the results are shown in Tables 1-2.

[0047] The analytical method described in Example 1 was modified in step 1 as follows: Normally developing zebrafish embryos at the cleavage stage (2 hpf) were randomly selected and placed in 90 mm culture dishes, exposed to 0.01% DMSO and PFOS (50, 500, 5000 ng / L) exposure solutions. Five replicates were set up for each concentration, with 100 embryos per replicate. Exposure was continuous from 2 hpf to 120 hpf. Throughout the exposure, the culture dishes were placed in a biochemical incubator at 28 ± 0.5 ℃ with a light-dark ratio of 14 h: 10 h. The exposure solution was prepared and changed daily. During the entire exposure period, 24 hpf and 72 hpf zebrafish embryos / larvae were selected for sample collection and body and yolk separation. Five replicates were set up for each concentration. Sixteen Embryo, Body, or Yolk pellets were collected from each replicate sample for lipidomics analysis.

[0048] Analysis revealed that lipidomic analysis of zebrafish embryos / larvae exposed to PFOS at 24 hpf and 72 hpf showed the following lipid counts: 1875 lipids were detected in the Body and Yolk lipid group of 24 hpf zebrafish embryos, and 1238 lipids were detected in the Embryo lipid group; 1910 lipids were detected in the Body and Yolk lipid group of 72 hpf zebrafish larvae, and 1440 lipids were detected in the Embryo lipid group. Differential lipid results showed that 97 differentially expressed lipids (19 classes) were detected in the Body & Yolk lipid group at 50 ng / L PFOS exposure, and 10 differentially expressed lipids (4 classes) were detected in the Embryo lipid group; 106 differentially expressed lipids (20 classes) were detected in the Body & Yolk lipid group at 500 ng / L PFOS exposure, and 12 differentially expressed lipids (6 classes) were detected in the Embryo lipid group at 5000 ng / L PFOS exposure; and... The Body & Yolk lipidome detected 145 differentially expressed lipids, categorized into 22 classes, in the PFOS exposure group, while the Embryo lipidome detected 14 differentially expressed lipids, categorized into 7 classes. See Table 1 for details. This demonstrates that the proposed method detects a large number and variety of lipids, exhibiting high sensitivity and the ability to detect subtle changes and key lipid molecules that are otherwise undetectable in the overall sample. Furthermore, this embodiment shows that compared to the time-consuming and laborious step-by-step processing methods in existing technologies, this method significantly overcomes the efficiency bottleneck of sample preparation. This not only ensures the rapid acquisition of large-scale samples but also makes it particularly suitable for dose gradient screening studies in environmental toxicology assessment. Thanks to its high-throughput characteristics, it can efficiently construct concentration gradient experiments with sufficient statistical effect.

[0049] Table 1. Total lipid counts detected in whole (Embryo) and partition (Body & Yolk) zebrafish after PFOS exposure.

[0050] Table 2. Differences in lipid detection between whole (Embryo) and local (Body & Yolk) zebrafish after exposure to environmental PFOS concentrations.

[0051] To investigate the effects of perfluorooctane sulfonate (PFOA) exposure on lipid metabolism in zebrafish embryos using spatially resolved lipidomics, tests were conducted, and the results are shown in Tables 3-4.

[0052] This study used spatially resolved lipidomics to reveal the effects of perfluorooctanoic acid (PFOA) exposure on lipid metabolism in zebrafish embryos. The analytical method described in Example 1 was modified by changing step 1. Specifically, normally developing cleavage-stage embryos (2 hpf) were randomly selected and placed in 90 mm culture dishes, exposed to 0.01% DMSO and PFOA (50, 500, 5000 ng / L) solutions. Five replicates were set up for each concentration, with 100 embryos per replicate. Exposure was continuous from 2 hpf to 120 hpf. Throughout the exposure, the culture dishes were placed in a biochemical incubator at 28 ± 0.5 ℃ with a light-dark ratio of 14 h: 10 h. The exposure solution was prepared and changed daily. During the entire exposure period, 24 hpf and 72 hpf zebrafish embryos / larvae were selected for sample collection, and the body and yolk were separated. Five replicates were set up for each concentration, and 16 Body, Yolk, or Embryo pellets were collected from each replicate sample for lipidomics analysis.

[0053] Analysis of the experimental results revealed that lipidomic analysis of zebrafish embryos / larvae exposed to PFOA at 24 hpf and 72 hpf showed the following lipid counts: 1757 lipids were detected in the Body and Yolk lipid group of 24 hpf zebrafish embryos, and 998 lipids were detected in the Embryo lipid group; 2022 lipids were detected in the Body and Yolk lipid group of 72 hpf zebrafish larvae, and 1406 lipids were detected in the Embryo lipid group. Differential lipid results showed that in the 50 ng / L PFOA exposure group, 31 differentially expressed lipids (14 classes) were detected in the Body and Yolk lipid group, and 6 differentially expressed lipids (5 classes) were detected in the Embryo lipid group; in the 500 ng / L PFOA exposure group, 83 differentially expressed lipids (18 classes) were detected in the Body and Yolk lipid group, and 6 differentially expressed lipids (6 classes) were detected in the Embryo lipid group; and in the 5000 ng / L PFOA exposure group… The Body & Yolk lipidomic assay detected 179 differentially expressed lipids, categorized into 20 classes, in the PFOS exposure group, while the Embryo lipidomic assay detected 33 differentially expressed lipids, categorized into 6 classes. The results are shown in Tables 3-4. This demonstrates that the proposed method detects a large number and variety of lipids, exhibiting high sensitivity and the ability to detect subtle changes and key lipid molecules that are otherwise undetectable in the overall sample. Furthermore, this example demonstrates that the method not only rapidly acquires large quantities of homogeneous samples but also, due to its superior throughput, is particularly crucial in environmental toxicology assessments requiring multiple concentration gradients, laying a solid foundation for obtaining reliable, high-resolution dose-response data.

[0054] Table 3. Total lipid counts detected in whole (Embryo) and partition (Body & Yolk) zebrafish after PFOA exposure.

[0055] Table 4. Differences in lipid detection between the whole (Embryo) and local (Body & Yolk) zebrafish after exposure to environmental PFOA concentrations.

[0056] Therefore, this application provides a spatially resolved lipidomics method based on zebrafish, comprising: placing zebrafish washed with PBS buffer on a culture dish covered with agarose gel, physically demetabolizing them under a stereomicroscope to obtain demetabolized zebrafish; adjusting the body position of the demetabolized zebrafish so that their tails face down, yolk sacs face right, lying on their sides in a regular arrangement, and sequentially aspirating the yolk sacs with a pipette to obtain secondary-treated zebrafish; collecting the body parts of the secondary-treated zebrafish, aspirating PBS buffer into a tube, vortexing at low speed for 30-60 s, washing the cleaned body parts with PBS buffer to obtain the body parts and yolk sacs, collecting them separately, and storing them in liquid nitrogen; after pretreatment of the body parts and yolk sacs, performing lipidomics detection on the samples using ultra-high performance liquid chromatography tandem quadrupole electrostatic field orbital trap high-resolution mass spectrometry, and performing data analysis. The method provided in this application detects a large number and variety of lipids, has high sensitivity, and can detect subtle changes and key lipid molecules that cannot be identified in the whole sample.

[0057] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0058] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. A spatially resolved lipidomics method based on zebrafish, characterized in that, The method includes: Zebrafish washed with PBS buffer were placed on agarose gel and physically demetabolized under a stereomicroscope to obtain demetabolized zebrafish. Adjust the body position of the zebrafish after molting so that its tail is facing down, its yolk sac is facing right, it is lying on its side, and its yolk sac is arranged regularly. Extract the yolk sac to obtain a secondary-processed zebrafish. Collect the zebrafish that have undergone secondary treatment, add them to PBS buffer and vortex them, wash the cleaned body with PBS buffer to obtain the body and yolk, collect them separately and store them in liquid nitrogen; The torso and egg yolk were pretreated, and the samples were then subjected to lipidomics detection using ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbital trap high-resolution mass spectrometry for data analysis.

2. The spatially resolved lipidomics method based on zebrafish according to claim 1, characterized in that, The zebrafish include zebrafish embryos with a 1-23 hpf, zebrafish embryos with a 24-72 hpf, and zebrafish larvae with a 72 hpf or higher.

3. The spatially resolved lipidomics method based on zebrafish according to claim 1, characterized in that, If the zebrafish are zebrafish larvae with a growth rate of 72 hpf or higher, after washing with PBS buffer, the zebrafish are placed on an agarose gel and placed under a stereomicroscope. No physical stripping step is required.

4. The spatially resolved lipidomics method based on zebrafish according to claim 1, characterized in that, If the zebrafish is a 1-23 hpf zebrafish embryo, the germ cells of the demembranous zebrafish are torn apart from the yolk to obtain the trunk and yolk, which are collected separately and stored in liquid nitrogen.

5. The spatially resolved lipidomics method based on zebrafish according to claim 1, characterized in that, Pretreatment of the trunk and yolk includes: The obtained torso and egg yolk were extracted separately with an extractant, centrifuged, concentrated under vacuum, reconstituted with a reconstituted solution, and centrifuged again to complete the pretreatment.

6. The spatially resolved lipidomics method based on zebrafish according to claim 5, characterized in that, The extractant is a mixed solution of chloroform and methanol in a volume ratio of 2:1, with 0.01% as an internal standard for lipidomics analysis; The complex solution is a mixture of isopropanol, acetonitrile, and water in a volume ratio of isopropanol:acetonitrile:water = 6:13:

1.

7. The spatially resolved lipidomics method based on zebrafish according to claim 1, characterized in that, After pretreatment of the torso and egg yolk, lipidomics detection of the samples was performed using ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbital trap high-resolution mass spectrometry, including: The liquid phase conditions were as follows: Mobile phase A: a mixed solution of 40% water and 60% acetonitrile; Mobile phase B: a mixed solution of 90% isopropanol and 10% acetonitrile; In positive ion mode, 0.1% formic acid and 10 mmol·L⁻¹ were added to both mobile phases. -1 Ammonium formate, in negative ion mode, 10 mmol·L⁻¹ was added to both mobile phases. -1 Ammonium acetate.

8. The spatially resolved lipidomics method based on zebrafish according to claim 1, characterized in that, After pretreatment of the torso and egg yolk, lipidomics detection of the samples was performed using ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbital trap high-resolution mass spectrometry, including: Gradient elution: 0 min, 37% B; 0–1.5 min, 37% B; 1.5–15.6 min, B linearly increases to 85%; 15.6–18 min, 97% B; 18–18.2 min, returns to the initial mobile phase ratio of 37% B; 18.2–20 min, 37% B.

9. The spatially resolved lipidomics method based on zebrafish according to claim 1, characterized in that, After pretreatment of the torso and egg yolk, lipidomics detection of the samples was performed using ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbital trap high-resolution mass spectrometry, including: An electrospray ionization source was used, scanning in both positive and negative ion modes; the scanning range was m / z 200–1700; the spray voltage was 3.5 kV for positive ion mode and 2.8 kV for negative ion mode; the sheath gas pressure was 35 AU; the auxiliary gas pressure was 15 AU; the backup gas pressure was 0 AU; the capillary temperature was 300 ℃; the ion transmission tube temperature was 320 ℃; the scanning mode was full scan, and the resolution was set to 120,000 full width at half maximum (FWHM); data-dependent mass spectrometry (ddMS) was used. 2 ), and fragment detection experiments were conducted on the parent ion.

10. The spatially resolved lipidomics method based on zebrafish according to claim 1, characterized in that, The data analysis includes: The test data were compared and analyzed with MS / MS fragment maps, mzVault, and MassList database information to identify lipid metabolites and determine the number and types of lipids.

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