CN112098503B - Method for in-vivo lipid three-dimensional mass spectrometry imaging based on integral zebra fish model - Google Patents
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Abstract
The invention belongs to the technical field of mass spectrometry detection, and discloses a method for three-dimensional mass spectrometry imaging of lipids in a whole zebra fish model. The invention establishes a method for three-dimensional mass spectrometry imaging of lipids in a whole zebra fish model, and applies the method to research of a zebra fish C1 type Niman Peak disease model, and results show that fifteen lipids with obvious spatial distribution differences exist in different organs of the zebra fish. The three-dimensional MALDI mass spectrum imaging method established by the invention does not need complex sample pretreatment, can detect various compounds in a tissue sample at the same time, can ensure the integrity of a selected tissue and the multidimensional visual effect, further intuitively and comprehensively obtain the spatial distribution difference of an experimental group and a control group, and has the advantages of high integrity and high chemical specificity.
Description
Technical Field
The invention relates to the technical field of mass spectrometry detection, in particular to a method for three-dimensional mass spectrometry imaging of lipids in a whole zebra fish model.
Background
MALDI mass spectrum imaging technology is a novel technology for visual analysis and detection, combines the component identification capability of mass spectrum with computer-aided image visualization technology, and can realize in-situ imaging analysis of various molecules and metabolites thereof such as small molecular compounds, polypeptides, proteins, nucleic acids, lipids and the like by adopting different matrixes at the tissue, cell or subcellular level. The method can realize in-situ qualitative and quantitative analysis of various molecules in the tissue sample without complex pretreatment, and draw out two-dimensional or three-dimensional spatial distribution diagrams of the molecules, and has the advantages of high sensitivity, high analysis speed and high chemical specificity. In recent years, the MALDI mass spectrometry imaging technology has made great progress, has been widely accepted as an effective tool for detecting and identifying various complex molecules in biological samples, and is widely applied to research fields such as life, biological medicine, environment and the like.
The application of the MALDI mass spectrometry imaging technology is mostly research on two-dimensional MALDI mass spectrometry imaging, but the two-dimensional MALDI mass spectrometry imaging method can only provide mass spectrometry imaging information of one section of a sample, and because a single section hardly reflects the integrity of the whole organ of a model, some important molecular information related to diseases is likely to be lost. While three-dimensional MALDI mass spectrometry imaging is a method of constructing three-dimensional images of multiple analytes in a target organ by taking serial slice mass spectrometry images of a sample and stacking them by specialized data processing software SCiLS. The method provides wider spatial information for the target molecules through deep sectional views, so that the spatial distribution of the target compounds in the animal model body can be reflected more truly and completely. There are some applications in three-dimensional MALDI mass spectrometry imaging, but it is limited to single organ or tissue studies, and many diseases involve multiple tissues or organs, such as NPC1 disease. The disease is autosomal recessive inherited lysosomal lipid storage disease, which accounts for 95% of NPC cases, and in recent years, the disease has been mainly studied on abnormal lipid metabolites in NPC1 disease individuals by constructing various animal models and using biological samples such as body fluid, single organ and the like, but the disease involves a plurality of organs such as viscera and nervous system, and the patient has symptoms such as spleen enlargement, dystonia, ataxia, mental retardation and the like. Therefore, if the three-dimensional imaging analysis can be carried out on the whole NPC1 animal, the pathological changes of different organs in the disease development process can be more comprehensively known.
Disclosure of Invention
In view of this, the present invention provides methods for three-dimensional mass spectrometry imaging of lipids in vivo based on whole zebra fish models. The three-dimensional MALDI mass spectrum imaging method established by the invention does not need complex sample pretreatment, can detect various compounds in a tissue sample at the same time, can ensure the integrity of a selected tissue and the multidimensional visual effect, further intuitively and comprehensively obtain the spatial distribution difference of an experimental group and a control group, and has the advantages of high integrity and high chemical specificity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for three-dimensional mass spectrometry imaging of lipids in a zebra fish model, which comprises the following steps:
step 1: zebra fish (Niemann-Pick C1, NPC 1) with homozygote of C1 type Niemann-Pick -/- ) And Wild Type (Wild Type, WT) zebra fish, after embedding, NPC1 was obtained, respectively -/- Frozen tissue sections of group and WT group, post-matrix deposition MALDI mass spectrometry acquisitionImaging data;
step 2: through SCiLS treatment, the X axis and the Y axis are adjusted to enable the frozen tissue slices to be accurately stacked according to the original shape of organisms:
step 3: comparative NPC1 -/- Three-dimensional imaging results of mass to charge ratios (m/z) detected by frozen tissue sections of the group and the WT group, obtaining m/z values with spatially distributed differences; and collecting MALDI secondary fragment information thereof;
step 4: searching a lipidomic mass spectrum database, and combining the MALDI secondary fragment information to obtain the lipids corresponding to the m/z values of the spatial distribution differences.
In some embodiments of the invention, the embedding in step 1 comprises: zebra fish (Niemann-Pick C1, NPC 1) with homozygote of C1 type Niemann-Pick -/- ) And Wild Type (Wild Type, WT) zebra fish were embedded with the whole zebra fish with an embedding medium and allowed to solidify at-80 ℃; a frozen tissue slice sample with a thickness of 14 μm is taken every 56 μm at-20 ℃ and adhered to an ITO plate, ensuring that the frozen tissue slice taken includes all whole organ tissues of zebra fish; NPC1 -/- At least 20 of the frozen tissue sections were taken from each of the group and WT.
In some embodiments of the invention, the embedding agent comprises a mixture of 10% sodium carboxymethyl cellulose and 10% gelatin.
In some embodiments of the invention, the substrate used for the substrate deposition in step 1 is N- (1-naphthyl) ethylenediamine dihydrochloride at a concentration of 7mg/mL and 50% methanol.
In some embodiments of the invention, the conditions for the deposition of the substrate in step 1 include: 35% of spraying power; modulating to 20%; the deposition time was 1.0s; incubation time was 15s; the drying time was 60s.
In some embodiments of the invention, the sampling conditions of the MALDI mass spectrometry imaging in step 1 comprise: the mass detection range is 400-1000Da; the detection voltage is 3.0×2810v; the spatial resolution is 50um; the ion source uses pulse laser, and the laser energy is 70%; the ion mode is a negative ion emission mode, and the ion type is [ M-H ]] - 。
In some embodiments of the invention, the search conditions in step 4 include: the deviation of molecular weight is +/-0.1 Da, and the anion mode is selected from [ M-H ]] - 。
In some embodiments of the invention, the distribution differences are present in the brain, eyes, spine, heart and intestines.
In some embodiments of the invention, the lipid comprises one or more of lysophosphatidylethanolamine, lysophosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol;
the lysophosphatidylethanolamine comprises C 21 H 44 NO 7 P or C 25 H 44 NO 7 P；
The lysophosphatidylinositol includes C 27 H 53 O 12 P；
The phosphatidylethanolamine comprises C 38 H 73 O 8 NP、C 43 H 77 O 7 NP、C 43 H 81 O 8 NP、C 45 H 79 O 8 NP、C 47 H 72 NO 8 P or C 47 H 80 NO 8 One or more of P;
the phosphatidylglycerol comprises C 47 H 80 NO 8 P or C 50 H 75 O 10 P；
The phosphatidylinositol comprises C 45 H 79 O 13 P、C 47 H 80 O 12 P、C 46 H 80 O 13 P or C 47 H 82 O 13 One or more of P.
Based on the research, the invention also provides a three-dimensional mass spectrum of the lipid in the zebra fish model body prepared by the method.
The invention provides a method for three-dimensional mass spectrometry imaging of lipid in a whole zebra fish model. The method comprises the steps of taking continuous frozen section tissue samples of different cross sections of the same zebra fish model, and placing the tissue samples on an Indium Tin Oxide (ITO) glass plate; then, performing matrix deposition, and performing matrix assisted laser desorption ionization (Matrix Assisted LaserDesorption Ionization, MALDI) mass spectrometry imaging analysis on the serial slice samples after the matrix deposition is completed; then reconstructing a three-dimensional mass spectrum imaging pattern by using professional data processing software SCiLS; lipids were identified by searching the lipidomic mass spectrometry database in combination with secondary fragmentation information. The invention establishes a method for three-dimensional mass spectrometry imaging of lipids in a whole zebra fish model, and applies the method to research of a zebra fish C1 type Niemann Pick (NPC 1) disease model, and results show that fifteen lipids with obvious spatial distribution differences exist in different organs of the zebra fish. The three-dimensional MALDI mass spectrum imaging method established by the invention does not need complex sample pretreatment, can detect various compounds in a tissue sample at the same time, can ensure the integrity of a selected tissue and the multidimensional visual effect, further intuitively and comprehensively obtain the spatial distribution difference of an experimental group and a control group, and has the advantages of high integrity and high chemical specificity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a schematic diagram of a method for three-dimensional mass spectrometry imaging of lipid in zebra fish bodies;
FIG. 2 shows a comparison of two-dimensional MALDI mass spectrometry imaging with three-dimensional MALDI mass spectrometry imaging;
FIG. 3 shows NPC1 -/- Three-dimensional MALDI mass spectrometry imaging lipid spatial distribution contrast plots for group and WT group.
Detailed Description
The invention discloses a method for three-dimensional mass spectrometry imaging of lipids in a whole zebra fish model, and a person skilled in the art can refer to the content of the invention to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
A method for three-dimensional mass spectrometry imaging of lipids in a whole zebra fish model comprises the following specific steps:
s1.C1. Niemann-Pick homozygote (Niemann-Pick C1, NPC 1) -/- ) And Wild Type (Wild Type, WT) zebra fish, embedding the whole zebra fish with an embedding agent, and placing in a refrigerator at-80 ℃ for solidification; then preparing frozen section samples, taking a slice of tissue section sample with the thickness of 14 μm at intervals of 56 μm in a frozen microtome at-20 ℃ and adhering the slice of tissue section sample to an ITO plate, NPC1 -/- Taking 20 pieces of the group and the WT group respectively, and ensuring that the taken tissue slices comprise all whole organ tissues of the zebra fish; placing the ITO plates adhered with the tissue slices in a vacuum dryer for 30min, and then performing matrix deposition, wherein each plate needs about 6mL; and after the matrix deposition is finished, carrying out MALDI mass spectrum imaging data acquisition on the sample.
S2, using professional data processing software SCiLS, and accurately stacking 20 tissue slices according to the original shape of the organism by adjusting the x-axis and the y-axis, so that the tissue slices approximately represent the whole animal model.
S3, comparing NPC1 -/- Three-dimensional imaging results of mass to charge ratios (m/z) detected by the group and the WT group, find m/z with spatial distribution differences; and the MALDI secondary fragment information is acquired.
S4, determining molecular formulas and lipid component names corresponding to the m/z values of the differences by searching a lipidomic mass spectrum database and combining MALDI secondary fragment information.
In some embodiments, the ion source for MALDI mass spectrometry uses pulsed laser to generate single charge ions, the mass spectrum peaks in the mass spectrum have a one-to-one correspondence with the mass numbers of the components of the sample, the ionization efficiency and the sensitivity are both high, and the three-dimensional MALDI mass spectrometry can comprehensively understand the spatial distribution difference of molecules in the tissue sample in a larger range than the two-dimensional MALDI mass spectrometry.
In some embodiments, the embedding agent described in step S1 is a mixture of 10% sodium carboxymethylcellulose +10% gelatin.
In some embodiments, the substrate described in step S1 is N- (1-naphthyl) ethylenediamine dihydrochloride (N- (1-naphthalenyl) ethylidedi-amine dihydrochloride, NEDC) at a ratio of 7mg/mL,50% methanol.
In some embodiments, the sampling conditions for mass spectrometry imaging described in step S1 are: the mass detection range is 400-1000Da; the detection voltage is 3.0×2810v; the spatial resolution is 50um; the laser energy is 70%; the ion mode is a negative ion emission mode, and the ion type is [ M-H ]] - 。
Zebra fish has been receiving more and more attention in recent years as an emerging research model due to small living space requirement, low maintenance cost, rapid production cycle and rapid growth, and has been widely applied to the construction of clinical disease models due to its gene sequences and organ systems similar to those of humans. At present, a plurality of reports of related lipid imaging researches on zebra fish by utilizing a two-dimensional mass spectrum imaging technology prove that the zebra fish has good development prospect as an ideal vertebrate model. The invention establishes a method for three-dimensional mass spectrometry imaging of lipid in a whole zebra fish model body and uses NPC1 -/- Zebra fish is taken as a study object to explore the three-dimensional spatial distribution of lipids in the zebra fish.
Compared with the prior art, the invention has the following beneficial effects:
1. compared with two-dimensional MALDI mass spectrometry imaging, the three-dimensional MALDI mass spectrometry imaging technology established by the invention ensures the integral biological characteristics of an animal model by acquiring continuous slices of a sample; the information of one dimension (z axis) is added, so that wider spatial information is provided for target molecules; the spatial distribution of the lipid in the disease model body can be obtained more intuitively and truly by utilizing the multidimensional visual effect.
2. The invention researches the spatial distribution situation of the lipid in the whole body of the whole zebra fish model, and can more accurately and comprehensively reflect the influence of the compounds on all organs of the whole body of an affected individual in the disease development process.
In the method for three-dimensional mass spectrometry imaging of lipids in the whole zebra fish model, raw materials and reagents used can be purchased from the market.
The lipidomic mass spectrum database in the invention is lipidresearch 4.0.
The invention is further illustrated by the following examples:
the following operations are performed according to the method based on the in vivo lipid three-dimensional mass spectrometry imaging of the whole zebra fish model shown in fig. 1:
example 1 acquisition of Whole zebra fish Mass Spectrometry imaging data
Frozen section sample preparation: NPC1 -/- After euthanasia of adult zebra fish of group and WT group, the whole zebra fish was embedded with an embedding medium and placed in a refrigerator at-80 ℃ until it solidified; then preparing frozen section samples, taking a slice of tissue section sample with the thickness of 14 μm at intervals of 56 μm in a frozen microtome at-20 ℃ and adhering the slice of tissue section sample to an ITO plate, NPC1 -/- The group and the WT group were each 20 pieces. The ITO plates with the tissue sections attached were placed in a vacuum oven for 30min and NEDC matrix deposition was performed with approximately 6mL of each plate.
Matrix deposition instrument: matrix deposition apparatus (Imageprep, bruker company). Deposition conditions: 35% of spraying power; modulating to 20%; the deposition time was 1.0s; incubation time was 15s; the drying time was 60s.
Mass spectrometry instrument: matrix assisted laser desorption ionization source (Matrix Assisted LaserDesorption Ionization, MALDI, bruker). Mass spectrometry conditions: the mass detection range is 400-1000Da; the detection voltage is 3.0×2810v; the laser energy is 70%; the spatial resolution is 50um; the ion mode is a negative ion reflection mode; the ion type is [ M-H ]] - 。
Example 2 construction of three-dimensional MALDI Mass Spectrometry images
Mass spectrometry imaging data were imported into the sciLS Lab 2016b (Bremen, germany) Premium 3D for processing, and 20 tissue sections could be accurately stacked according to the original morphology of the organism by adjusting the x and y axes to approximate a whole animal model. Normalizing by a total ion flow method, setting the m/z interval to be +/-0.1 Da, and processing all MALDI mass spectrum images by using a weak denoising mode so as to construct a mass spectrum image of the lipid at the original position of the tissue.
As shown in fig. 2: fig. 2A, 2B, and 2C are two-dimensional MALDI mass spectrometry images distributed on the spine (m/z is 452.324), brain (m/z 790.854), and intestine (m/z 867.534), respectively, with the largest cross-sections of the three organs located on the 11 th, 17 th, and 10 th slices, respectively, and fig. 2D, 2E, and 2F are three-dimensional MALDI mass spectrometry images of the ions in the corresponding organs, respectively. It can be seen from fig. 2A, 2B, and 2C that the largest cross-sections of different organs are located on different slices, so that the method of two-dimensional mass spectrometry imaging using a single cross-section is difficult to embody the integrity of all organs of the model, and may thus lose some important molecular information related to the disease. And the three-dimensional mass spectrum imaging images formed by stacking the serial sections are shown in fig. 2D, 2E and 2F, so that the integral biological characteristics of the animal model can be completely displayed. As can be seen by comparing fig. 2A and fig. 2F, the same organ cannot be fully represented on a single cross section. Therefore, compared with the two-dimensional MALDI mass spectrometry imaging, the three-dimensional MALDI mass spectrometry imaging method can more comprehensively display the spatial distribution condition of the lipid in each organ, so that the pathological changes of different organs in the disease development process can be more truly known.
Example 3 screening of differential lipids in zebra fish NPC1 disease models
Embedded euthanized NPC1 -/- Whole zebra fish from group and WT group, frozen section tissue samples were obtained and subjected to matrix deposition as in example 1, for NPC1, respectively -/- All slice samples of the group and WT group were subjected to MALDI mass spectrometry imaging, followed by reconstruction of the three-dimensional MALDI pattern according to the method of example 2. Fifteen different lipid m/z are obtained through screening, detected difference m/z values are input into a lipidomic mass spectrum database of Lipidseal 4.0 for searching, and are compared with the secondary fragment information to determine molecular formulas corresponding to the difference m/z values and lipid names corresponding to the molecular formulas. Wherein the search conditions are: the deviation of molecular weight is +/-0.1 Da, and the anion mode is selected from [ M-H ]] - . The results are shown in Table 1, where these differential lipids are distributed predominantly in the brain, eyes, spine, heart and intestine.
TABLE 1 NPC1 -/- Identification of differential lipid in group and WT group zebra fish
Three lipids, namely lysophosphatidylethanolamine LPE (16:0), phosphatidylethanolamine PE (18:0/22:5) and phosphatidylinositol PI (16:0/20:4), distributed in the spinal column, brain and intestinal tract, respectively, are exemplified in NPC1 -/- There was a significant difference in the distribution in the group and WT. As shown in fig. 3 a-c: lysophosphatidylethanolamine is distributed in the spine (FIG. 3 a), which is found in NPC1 -/- The content of the group is obviously less than that of the WT group; phosphatidylethanolamine is distributed in brain tissue (FIG. 3 b), which is the case in NPC1 -/- The content of the group is obviously more than that of the WT group; phosphatidylinositol is mainly distributed in the intestinal tract (FIG. 3 c), which is in NPC1 -/- The content of the group is significantly more than that of the WT group.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. The method for three-dimensional mass spectrometry imaging of the lipid in the zebra fish model is characterized by comprising the following steps of:
step 1: taking C1 type Niman Peak homozygote zebra fish and wild zebra fish, embedding, and respectively obtaining NPC1 -/- Frozen tissue sections of the group and the WT group, and MALDI mass spectrometry imaging data are acquired after matrix deposition;
step 2: through SCiLS treatment, the x and y axes are adjusted to accurately stack the frozen tissue slices according to the original biological morphology:
step 3: comparative NPC1 -/- Mass to charge ratio m/z detected by frozen tissue sections of group and WT groupObtaining m/z values with spatial distribution differences; and collecting MALDI secondary fragment information thereof;
step 4: searching a lipidomic mass spectrum database, and combining the MALDI secondary fragment information to obtain lipids corresponding to m/z values of the spatial distribution differences;
the embedding in step 1 comprises: embedding whole zebra fish with embedding medium of C1 type Niman Peak homozygote zebra fish and wild zebra fish, and solidifying at-80deg.C; a frozen tissue slice sample with a thickness of 14 μm is taken every 56 μm at-20 ℃ and adhered to an ITO plate, ensuring that the frozen tissue slice taken includes all whole organ tissues of zebra fish; NPC1 -/- At least 20 frozen tissue sections from each of the group and WT;
the substrate adopted in the substrate deposition in the step 1 is N- (1-naphthyl) ethylenediamine dihydrochloride with the concentration of 7mg/mL and 50% methanol;
the conditions for the deposition of the substrate in step 1 include: the spraying power is 35%; modulating to 20%; deposition time was 1.0s; incubation time was 15s; the drying time was 60 s;
the sampling conditions of the MALDI mass spectrometry imaging in step 1 include: the quality detection range is 400-1000Da; the detection voltage is 3.0×2810V; the spatial resolution is 50um; the ion source uses pulse laser, and the laser energy is 70%; the ion mode is a negative ion reflection mode, and the ion type is [ M-H ]] - ；
The lipid comprises one or more of lysophosphatidylethanolamine, lysophosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol;
the lysophosphatidylethanolamine comprises C 21 H 44 NO 7 P or C 25 H 44 NO 7 P；
The lysophosphatidylinositol includes C 27 H 53 O 12 P；
The phosphatidylethanolamine comprises C 38 H 73 O 8 NP、C 43 H 77 O 7 NP、C 43 H 81 O 8 NP、C 45 H 79 O 8 NP、C 47 H 72 NO 8 P or C 47 H 80 NO 8 One or more of P;
the phosphatidylglycerol comprises C 47 H 80 NO 8 P or C 50 H 75 O 10 P；
The phosphatidylinositol comprises C 45 H 79 O 13 P、C 47 H 80 O 12 P、C 46 H 80 O 13 P or C 47 H 82 O 13 One or more of P.
2. The method of claim 1, wherein the embedding medium comprises a mixture of 10% sodium carboxymethyl cellulose and 10% gelatin.
3. The method according to claim 1 or 2, wherein the search conditions in step 4 include: the molecular weight deviation is +/-0.1 Da, and the anion mode is selected from [ M-H ]] - 。
4. The method of claim 1, wherein the distribution differences are present in the brain, eye, spine, heart, and gut.
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