In-situ visual characterization method of oxidized lipid metabolites in biological tissue
Through the derivatization reaction of solid-state derivatized hydrogel HCD blocks in biological tissues, combined with AFADESI-MSI technology, the problems of low coverage of oxidized lipid metabolites and excessive reagents in the prior art are solved, and efficient and accurate identification effect is achieved.
Patent Information
- Application Number
- CN202411851353.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-16
- Publication Date
- 2025-03-25
AI Technical Summary
The prior art has problems of low coverage and excessive derivatization reagents when visualizing oxidized lipid metabolites in biological tissues, making it difficult to achieve efficient and accurate identification.
A solid derivatized hydrogel HCD block was used to prepare a 0.5-2% aqueous gel solution by combining agarose and melarazine, and a derivatization reaction was carried out at 4-25°C for 0.5-2.5 hours, and the analysis was performed in combination with AFADESI-MSI technology.
The high coverage and accurate identification of 326 oxidized lipid metabolites in the overall section samples of zebrafish adult fish was achieved. Compared with other methods, the conditions of 4°C are more friendly, the amount of derivatization reagents is used, and the coverage is higher.
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Figure CN119688813A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioanalytical chemistry, and in particular relates to an in-situ visual characterization method for oxidized lipid metabolites in biological tissues. Background Art
[0002] Mass spectrometry imaging (MSI) can provide spatial distribution, relative abundance, and structural information of multiple types of molecules in biological tissue samples. Although MSI has a higher detection coverage for biomolecules than other types of chemical imaging techniques, in situ characterization is still challenging due to the low abundance of some compounds or the structure that is not easily ionized. Therefore, more and more research focuses on the development of tissue chemical derivatization (OTCD) methods.
[0003] On the one hand, some studies have focused on the synthesis or selection of derivatization reagents. The common Girard reagent can introduce positively charged quaternary amine groups into carbonyl compounds, thereby improving the sensitivity of MSI analysis of neutral corticosteroids, cholesterol, exogenous steroid drugs, and terpenoid plant hormones. Fatty aldehydes (FAL) and oxygenated fatty acids (oxo-FA) are typical lipid metabolites that require derivatization for mass spectrometry detection, both of which have important physiological and pathological significance.
[0004] On the other hand, some studies focus on developing new methods for coating derivatization reagents. Different coating methods can affect the efficiency of OTCD and thus the results of MSI. Spraying with a sprayer or inkjet printer is a common and simple method in most studies. The electrospray principle is also applied to the sprayer device to produce tiny and uniform particles. In addition, sublimation is a solvent-free coating method that is currently less used because its reaction efficiency is lower than that of derivatization reactions in the presence of solvents. However, the common limitation of the above methods is that they will lead to excessive derivatization reagents. A study has developed a general hydrogel-assisted chemical derivatization (HCD) method, using gelatin as the loading medium for the Girard P reagent. The excess derivatization reagent can be removed after the derivatization reaction is completed through a "fit-remove" operation. Summary of the invention
[0005] The present invention provides an in situ visualization characterization method for oxidized lipid metabolites in biological tissues, which achieves high coverage and accurate identification of 326 oxidized lipid metabolites in whole-body slice samples of adult zebrafish.
[0006] To achieve the above object, the present invention adopts the following technical solutions:
[0007] The present invention provides an in situ visual characterization method for oxidized lipid metabolites in biological tissues, the method comprising the following steps:
[0008] (1) Sample pretreatment: frozen sectioning of the histological samples to be tested;
[0009] (2) preparing a solid derivatized hydrogel HCD block: preparing a 0.5-2% gel aqueous solution of agarose, adding a derivatizing agent methyhydrazine with a final concentration of 0.5-5 mg / mL and a 0.5-1.5% formic acid solution to the gel aqueous solution, and freezing and solidifying the solution to obtain a solid derivatized hydrogel HCD block;
[0010] (3) Derivatization reaction: placing the solid derivatized hydrogel HCD block on the surface of the tissue slice and placing it at 4-25°C for 0.5-2.5h for derivatization reaction;
[0011] (4) After the derivatization reaction is completed, the derivatization product is analyzed using AFADESI-MSI technology, and the corresponding mass spectrometry imaging data is collected.
[0012] Furthermore, the pretreatment step in step (1) is: rapidly freezing the sample, and then preparing a tissue section with a thickness of 10-15 μm at the maximum radial surface of the sample tissue.
[0013] Furthermore, the pretreatment step is: rapidly freezing the sample, and then preparing a tissue section with a thickness of 12 μm at the maximum radial surface of the sample tissue.
[0014] Furthermore, the steps of preparing the solid-state derivatized hydrogel HCD block are: preparing a 1% gel aqueous solution with agarose, adding a derivatization reagent methyhydrazine with a final concentration of 2 mg / mL and a 1% formic acid solution to the gel aqueous solution to form a derivatized hydrogel solution; placing the solution in a mold to solidify at 4°C, and after demolding, a solid-state derivatized hydrogel HCD block is obtained.
[0015] Furthermore, the derivatization reagent melazone is composed of d0-melazone and heavy isotope-labeled d 20 -melazine, wherein the d0-melazine and heavy isotope labeled d 20 -The dosage ratio of melarizine is 0.5~1.5:0.5~1.5.
[0016] Further, the d0-melazine and heavy isotope labeled d 20 -Melarizine dosage ratio is 1:1.
[0017] Furthermore, the reaction temperature in step (3) is 4°C.
[0018] Furthermore, the reaction time in step (3) is 1.5 h.
[0019] Furthermore, the analysis method parameters in step (4) are set as follows: the volume ratio of the spray solvent MeOH / H2O is 8:2, and the flow rate is 7 μL / min; the spray gas is nitrogen, and the gas pressure is 0.6 MPa; the spray voltage is 3 kV; the temperature of the mass spectrometer ion transfer tube is 350°C; the pumping flow rate is 45 L / min; the movement speed of the three-dimensional electric platform in the x direction is 0.2 mm / s, and the step length in the y direction is 0.2 mm; the mass spectrometer acquisition mode is full scan, m / z 100-1000, positive ion mode, mass resolution is 120000, and automatic gain control AGC target is 1e 6 , the maximum injection time MIT is 100ms.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] The present invention is prepared by preparing a d0 / d containing 2 mg / mL concentration 20 -1% agarose hydrogel blocks of melazone were attached to zebrafish tissue sections for 1.5 hours at 4°C for derivatization. After AFADESI-MSI analysis, high coverage in situ visualization of 326 oxidized lipid metabolites in vivo was achieved. Compared with other methods of the same type, it is more efficient, 4°C conditions are more friendly to biological samples, the amount of derivatization reagents used is also less, and the coverage of oxidized lipid metabolites is higher. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is a diagram showing the effect of the hydrogel type on the detection results of representative oxidized lipid metabolites in Example 1;
[0023] Figure 2 Figure 1 is a graph showing the effects of derivatization temperature, time, and derivatization reagent concentration on the detection results of representative oxidized lipid metabolites in Example 1 (Figure A is an optimization graph of derivatization temperature and time; Figure B is a graph showing the effects of 25°C and 4°C on zebrafish whole tissue section detection; Figure C is a graph showing the optimization of derivatization reagent concentration);
[0024] Figure 3 The representative oxidized lipid metabolites in Example 1 are d0-melazine, d 20 -MSI diagram of melarizine labeled product; DETAILED DESCRIPTION
[0025] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.
[0026] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.
[0027] Example 1
[0028] 1. Reagents and Materials
[0029] The derivatization reagents melazone and d20-melazone were synthesized by Shenzhen Kesai Chemical Biotechnology Co., Ltd.; HPLC-grade methanol was purchased from Merck (Merck, Germany); HPLC-grade formic acid was purchased from Tedia (Fairfield, OH, USA); purified water was purchased from Wahaha (Hangzhou, China); standard compounds were purchased from Aladdin (St. Louis, MO, USA), including FAL 6:0 (C6H 12 O), FAL 7:0(C7H 14 O), FAL 8:0(C8H 16 O), FAL 9:0(C9H 18 O), FAL 11:0(C 11 H 22 O) and FAL 12:0(C 12 H 24 O). Prepare a 1 mg / mL stock solution with methanol, and then prepare a mixed standard solution with a concentration of 100 μg / mL.
[0030] 2. Zebrafish Sample Pretreatment
[0031] Wild AB adult zebrafish were purchased from Shandong Yixiyue Biotechnology Co., Ltd.
[0032] After starvation for 24 h, zebrafish were placed in an ice-water mixture, and the eye movements of zebrafish in the frozen water were observed. The fish were removed 2 min after the last eye movement. The surface moisture of the zebrafish was blotted with a paper towel, spread on tin foil, and placed in liquid nitrogen for rapid freezing. Then, the zebrafish frozen in liquid nitrogen was placed in a 3 cm × 3 cm silicone ice cube mold, water was added as an embedding agent, and the fish was frozen in a -80 ° C refrigerator. Before making frozen tissue sections of the samples, the embedded zebrafish was peeled off from the mold and fixed on the sample holder of the cryostat. 12 μm frozen tissue sections were prepared at the maximum radial surface of the zebrafish tissue. Tissue sections were used to visualize the oxidized lipid metabolites in zebrafish in situ. The sections needed to be completely dried in a vacuum desiccator before AFADESI-MSI analysis.
[0033] Preparation of tissue homogenate slices: Wild AB adult zebrafish tissue was taken and added with physiological saline at a ratio of 1:3 (g / mL), and prepared into tissue homogenate using a tissue cell disruptor. The homogenate was placed in a 1cm×1cm silicone ice cube mold and frozen in a -80℃ refrigerator. On a freezing microtome, 30μm uniform and reproducible tissue homogenate slices were prepared at -22℃. The tissue homogenate slices were used for experimental condition optimization. The slices needed to be completely dried in a vacuum desiccator before AFADESI-MSI analysis.
[0034] 3. In situ derivatization
[0035] Add pure water to the agarose solid to prepare a 1% agarose solution, heat and stir to fully dissolve it, and let it stand to expel bubbles. 20 -Melarzine derivatization reagent (melazine and heavy isotope labeled d 20 The derivatized hydrogel solution was transferred to a silicone mold (30×11×10 mm per grid). 3 , length × width × height), and cooled at 4°C until solidified, and then it becomes a HCD block after demoulding.
[0036] The HCD block was placed on the surface of the tissue section product prepared above and placed at 4°C for 1.5 h for derivatization reaction.
[0037] 4. AFADESI-MSI Analysis
[0038] After the derivatization reaction was completed, the samples were analyzed using the AFADESI-MSI platform and a Q-OT-qIT mass spectrometer (Orbitrap Fusion Lumos, Thermo Fisher Scientific, USA).
[0039] The parameters were set as follows: the spray solvent was MeOH / H2O (8:2, v / v), the flow rate was 7 μL / min; the spray gas was nitrogen, the pressure was 0.6 MPa; the spray voltage was 3 kV; the mass spectrometer ion transfer tube temperature was 350 °C; the pumping flow rate was 45 L / min; the movement speed of the three-dimensional electric platform in the x direction was 0.2 mm / s, and the step length in the y direction was 0.2 mm; the mass spectrometer acquisition mode was full scan, m / z 100-1000, positive ion mode, mass resolution was 120000, and automatic gain control (AGC target) was 1e 6 , the maximum injection time (MIT) is 100ms.
[0040] 5. Data processing
[0041] The raw data were imported into MassImager software for image reconstruction. After background subtraction and normalization, the region of interest (ROI) was selected to generate an average mass spectrum, which was converted into an ion list and exported to Markerview 1.2.1 (SCIEX) software for peak alignment and isotope removal. According to the derivatization reaction mechanism, the precise m / z of the original metabolite can be obtained from (MC 11 H 23 O7+H2O) or (MC 11 H3D 20 O7+H2O) calculated, where M is melazine or d 20- m / z of melazone derivatives; m / z of melazone or d 20- The m / z difference of melarizine derivatives was 20.0155. Carbonyl metabolites were identified by searching the LIPID MAPS database, with the m / z error tolerance set to 5 ppm.
[0042] Furthermore, MS / MS analysis was used to confirm the inferred derivatization products, where the characteristic daughter ions of the melarizine derivatization products were m / z 209.1509, d 20 The characteristic daughter ion of -melamine derivative product is m / z 224.2451.
[0043] Since there is no hydrogel-assisted in situ derivatization analysis method for frozen tissue samples of adult zebrafish in the early stage, the present invention investigates the effect of the type of hydrogel on the test results, and also investigates the effect of the derivatization reaction temperature, derivatization reaction time, and derivatization reagent concentration on the test results. The specific investigation conditions are as follows:
[0044] (1) Embedding medium type: 0.5% agarose, 1% agarose, 2% agarose, 15% gelatin, 20% gelatin;
[0045] (2) Derivatization reaction temperature: 4°C, 25°C;
[0046] (3) Derivatization reaction time: 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h;
[0047] (4) Derivatization reagent concentration: 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL;
[0048] In addition, 1% formic acid was added during the preparation of the hydrogel solution to provide an acidic environment for the derivatization reaction.
[0049] First, the present invention investigates two hydrogel materials, agarose and gelatin, and the detection effect of different proportion solutions on carbonyl lipid metabolites. The concentration of agarose solution is set to 0.5%-2%, and the concentration of gelatin is set to 15% and 20%, respectively. The results show that the strength of six standards, FAL 6:0, FAL 7:0, FAL 8:0, FAL 9:0, FAL 11:0 and FAL 12:0, decreases with the increase of the proportion of hydrogel solution, and agarose is a more suitable loading medium for melarizine. After 0.5% agarose HCD treatment, the intensity reaches the highest value, followed by 1% agarose. Considering that the difference in the derivatization effect between the two is not obvious, and the 1% agarose hydrogel has higher mechanical strength and is not easy to cause metabolite delocalization, the agarose hydrogel with a concentration of 1% is used as the most suitable hydrogel material.
[0050] Secondly, melarizine has strong nucleophilicity and can be derivatized under very mild conditions, such as room temperature. However, considering that 4°C is beneficial to the physical stability of the HCD block and the chemical stability of biological samples, the temperature parameters of 25°C and 4°C were investigated. On the other hand, under the premise that other parameters remain unchanged, the reaction time conditions were set to 0.5-2.5h for investigation. The results show that the optimal derivatization effect can be achieved for most targets within 1.5h, and the mass spectrometry response obtained under 25°C is slightly higher than that under 4°C, but the difference is not significant. Then, the zebrafish whole slices were derivatized and it was found that the metabolite coverage obtained at 4°C was more advantageous than that at 25°C, such as FAL5:1;O (m / z 336.2506), FAL5:2;O (m / z 334.2350), FAL6:2 (m / z 332.2557), FAL11:4 (m / z 398.3027) and FAL12:6 (m / z408.2807); while at 25°C, their imaging effects were poor or absent. In summary, 1.5h treatment at 4°C is the optimal derivatization reaction condition.
[0051] In addition, the HCD block containing 0.5-5.0 mg / mL of the derivatization reagent was investigated. The results showed that the response of the derivatization products of most target metabolites increased with the increase of the derivatization reagent concentration and reached the maximum value at 2 mg / mL. Therefore, the optimal derivatization reagent concentration is 2.0 mg / mL.
[0052] Furthermore, in order to verify d 20 Whether the addition of -melazine will have a negative impact on the reaction, the present invention also set up three experimental groups, using heavy isotope labeled d 20 -melamine and melahydrazine (the ratio of the two was 1:1) were mixed in HCD blocks as experimental group 1, melahydrazine alone was used as experimental group 2, and single d 20-HCD block of melarizine was used as experimental group 3. Based on the ion lists obtained in each experiment, possible derivative product ions can be obtained by comparative analysis. After AFADESI-MSI analysis, image reconstruction and screening of possible derivatization product ions, 355 and 384 possible derivatization product ions were obtained in experimental groups 2 and 3, respectively. According to the fixed m / z difference, 323 derivatization products can be matched between experimental groups 2 and 3. In the ion list obtained in experimental group 1, the isotope peak pairs can be matched directly according to the fixed m / z difference, and finally 326 possible derivatization products were found. The result is in good agreement with the result obtained using a single derivatization reagent. It can be concluded that the heavy isotope labeled reagent d 20 -Methylazine did not negatively affect the derivatization reaction.
[0053] Among the 326 possible derivatization products mentioned above, there are 188 FALs and 138 oxo-FAs. The specific detection information is shown in Tables 1 and 2 below. Figure 3 The representative oxidized lipid metabolites in tissue homogenate sections without and after derivatization are shown. 20 MSI image of the derivatization product labeled with -melazine. Although in tissue homogenate sections, it can still show the melazone-labeled derivative and d 20 The spatial distribution characteristics of the melazone-labeled derivatives of the above 326 metabolites were further analyzed by MS / MS. 20 -Melarizine-labeled derivatives were used for structural verification. The characteristic daughter ions m / z 209.1509, d 20 The characteristic daughter ion of -melamine labeled products was detected as m / z 224.2451. Therefore, the 326 derivatization products were reliable and accurate.
[0054] In summary, the method comprises preparing a 2 mg / mL concentration of d0 / d 20-1% agarose hydrogel block of melarizine was attached to zebrafish tissue sections at 4°C for 1.5h for derivatization. After AFADESI-MSI analysis, high coverage in situ visualization of 326 oxidized lipid metabolites in the body was achieved, of which 120 FALs and 83 oxo-FAs were not included in the LIPID MAPS database. It has been reported that other methods of the same type were used to derivatize three organ tissues of rat liver, kidney, and brain. It was necessary to use 50mg / mL of derivatization reagent and treat at room temperature for 2h, and finally 166 FALs and 100 oxo-FAs were obtained. Therefore, the method of the present invention is more efficient than the prior art, the 4°C condition is more friendly to biological samples, the amount of derivatization reagent is also less, and the coverage of oxidized lipid metabolites is higher.
[0055] Table 1d0 / d 20 - FALs metabolites labeled with melazone (metabolites not included in the LIPID MAPS database*)
[0056]
[0057]
[0058]
[0059]
[0060]
[0061]
[0062]
[0063]
[0064]
[0065] Table 2d0 / d 20 -Oxo-FAs metabolites labeled with methychloride (metabolites not included in the LIPID MAPS database*)
[0066]
[0067]
[0068]
[0069]
[0070]
[0071]
[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A method for in situ visualization characterization of oxidized lipid metabolites in biological tissues, characterized in that: The method comprises the following steps: (1) Sample pretreatment: frozen sectioning of the histological samples to be tested; (2) preparing a solid derivatized hydrogel HCD block: preparing a 0.5-2% gel aqueous solution of agarose, adding a derivatizing agent methyhydrazine with a final concentration of 0.5-5 mg / mL and a 0.5-1.5% formic acid solution to the gel aqueous solution, and freezing and solidifying the solution to obtain a solid derivatized hydrogel HCD block; (3) Derivatization reaction: placing the solid derivatized hydrogel HCD block on the surface of the tissue slice and placing it at 4-25°C for 0.5-2.5h for derivatization reaction; (4) After the derivatization reaction is completed, the derivatization product is analyzed using AFADESI-MSI technology, and the corresponding mass spectrometry imaging data is collected.
2. The characterization method according to claim 1, characterized in that The pretreatment step in step (1) is: rapidly freezing the sample, and then preparing a tissue section with a thickness of 10-15 μm at the maximum radial surface of the sample tissue.
3. The characterization method according to claim 2, characterized in that The pretreatment step is: rapidly freezing the sample, and then preparing a tissue section with a thickness of 12 μm at the maximum radial surface of the sample tissue.
4. The characterization method according to claim 1, characterized in that The steps of preparing the solid-state derivatized hydrogel HCD block are as follows: preparing a 1% gel aqueous solution with agarose, adding a derivatization reagent methyhydrazine with a final concentration of 2 mg / mL and a 1% formic acid solution to the gel aqueous solution to form a derivatized hydrogel solution; placing the derivatized hydrogel solution in a mold to solidify at 4° C., and obtaining a solid-state derivatized hydrogel HCD block after demolding.
5. The characterization method according to claim 1, characterized in that The derivatization reagent melazone is composed of d0-melazone and heavy isotope-labeled d 20 -melazine, wherein the d0-melazine and heavy isotope labeled d 20 -The dosage ratio of melarizine is 0.5~1.5:0.5~1.
5.
6. The characterization method according to claim 5, characterized in that The d0-melazine and heavy isotope labeled d 20 -Melarizine dosage ratio is 1:
1.
7. The characterization method according to claim 1, characterized in that: The reaction temperature in step (3) is 4°C.
8. The characterization method according to claim 1, characterized in that The reaction time in step (3) is 1.5 h.
9. The characterization method according to claim 1, characterized in that: The analysis method parameters in step (4) are set as follows: the volume ratio of the spray solvent MeOH / H2O is 8:2, and the flow rate is 7 μL / min; the spray gas is nitrogen, and the gas pressure is 0.6 MPa; the spray voltage is 3 kV; the temperature of the mass spectrometer ion transfer tube is 350°C; the pumping gas flow rate is 45 L / min; the movement speed of the three-dimensional electric platform in the x direction is 0.2 mm / s, and the step length in the y direction is 0.2 mm; the mass spectrometer acquisition mode is full scan, m / z 100-1000, positive ion mode, mass resolution is 120000, and automatic gain control AGC target is 1e 6 , the maximum injection time MIT is 100ms.