Method for MALDI (matrix-assisted laser desorption ionization) mass spectrometry imaging and space transcriptomics detection on same tissue slice

Abstract

The invention relates to a mass spectrometry imaging and space transcriptomics detection method, and discloses a method for MALDI (matrix-assisted laser desorption ionization) mass spectrometry imaging and space transcriptomics detection on the same tissue slice. According to the prepared durable MALDI-TOF target plate capable of loading the biological tissue on the space transcriptome chip, a high-sensitivity and visual mass spectrum imaging method for metabolic substances in the colon tissue on the space transcriptome chip is established by utilizing an MALDI mass spectrum imaging analysis method, and in addition, good space transcriptome data of the colon tissue on the same chip are obtained; the target plate overcomes the defects that a common target plate cannot fix a chip of the model, the thickness of a clamping groove is not matched with the thickness of the chip, and the clamping groove cannot meet the requirements that a coating on the surface of the chip cannot be damaged and a mass spectrum signal is captured on the space transcriptome chip, and has relatively high sensitivity; good space transcriptome and space metabolome data on the same tissue slice are obtained, and technical support is provided for conjoint analysis of space metabolomics and space transcriptomics.

Description

Technical Field

[0001] The invention relates to a method for mass spectrometry imaging and spatial transcriptomics detection, and in particular to a method for MALDI mass spectrometry imaging and spatial transcriptomics detection on the same tissue section. Background Art

[0002] Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF) is a method developed in recent years that can be used to characterize the spatial distribution characteristics of chemical components on biological tissues. This technology combines mass spectrometry and imaging technology. By dividing the sample surface into small areas, mass spectrometry is collected area by area to obtain chemical composition information of each area. After visualization, the distribution information of chemical components in the entire test area can be mapped, and the spatial positioning of the target analytical substance can be achieved. It has high sensitivity and high-quality distribution characteristics and is widely used in medical research, drug development, plant science, food science and other fields. This technology can deeply characterize a variety of metabolites such as metabolites and lipids in biological tissues, and can realize metabolic markers in lesion tissues and healthy tissues, thereby allowing the characterization of metabolic structures in biological tissues.

[0003] Spatial transcriptomics is a disruptive biological technology that combines biological technology with spatial imaging technology. It can simultaneously analyze gene expression and its spatial distribution in tissues under tissue morphology. This technology breaks the limitation of traditional transcriptomics that can only analyze the average expression of samples, allowing researchers to analyze the spatial distribution of gene expression at the single cell or micro-area level, and can fully understand the spatial expression law of genes at the tissue level, which helps to deeply reveal the spatiotemporal characteristics of gene regulation in biological systems and their functions and significance in biological processes. Introducing spatial transcriptomics into biological tissues will greatly reveal the interaction between cells at the transcriptional level in biological tissues. So far, there have been literature reports on the combined use of spatial metabolomics (MALDI-MSI) and spatial transcriptomics, but most of the literature's joint analysis uses adjacent biological tissue sections for separate detection and then joint analysis, but there are still errors caused by non-identical sections and it is technically difficult to obtain adjacent frozen section sections. In addition, there are literature reports that 10× chips have been applied to MALDI-MSI target plates for analysis. The current chips mainly include those from 10× Company, BGI, and Bio-Tech S1000, but the spatial transcriptome chip from Qingdao BGI has not been used in mass spectrometry imaging.

[0004] At present, the spatial transcriptome chip of BGI cannot be used on MALDI-MS because there is no MALDI-MS method developed for the spatial transcriptome chip of BGI. So far, most instruments mainly use metal target plates and metal target holders, which can be reused repeatedly. The common specifications are 75mm×25mm×1.1mm. However, there are no reports on target plates that can perform mass spectrometry imaging analysis on spatial transcriptome chips. The gap thickness of the slot design of ordinary metal target plates does not match the chip thickness. The slot design is difficult to meet the requirement that the chip surface coating cannot be damaged and is easy to cause the chip to break. In addition, the spatial transcriptome chip is made of silicon-based material, and the chip itself is not conductive. The poor conductivity of the surface of the spatial transcriptome chip causes poor mass spectrometry signals. Summary of the invention

[0005] The present invention aims at the requirements of high resolution, high sensitivity and high repeatability in MALDI-TOF detection, and at the same time, in order to overcome the problem that biological tissue on the spatial transcriptome chip cannot realize spatial metabolomics detection, a method for realizing MALDI mass spectrometry imaging and spatial transcriptomics detection on the same tissue section based on a novel target plate is provided, which realizes the visual representation of metabolites in colon tissue on the spatial transcriptome chip, has high sensitivity, and provides technical support for the joint analysis of spatial metabolomics and spatial transcriptomics.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for MALDI mass spectrometry imaging and spatial transcriptomics detection on the same tissue section comprises the following steps:

[0008] (1) Sample preparation: The sample tissue is embedded in OCT embedding medium, then sliced ​​using a freezing microtome and attached to a spatial transcriptome chip and conductive glass to obtain tissue slices, which are then vacuum dried;

[0009] (2) Matrix spraying: The sample tissue after vacuum drying is subjected to matrix spraying, wherein the matrix is ​​DAN / HCCA / DHB matrix, and tissue sections sprayed with the matrix are obtained;

[0010] (3) Tissue loading: The colon tissue sprayed with the matrix on the conductive glass was loaded onto a common metal target holder; the colon tissue sprayed with the matrix on the spatial transcriptome chip (1 cm×1 cm) was loaded onto the corresponding groove of the metal target plate. The loading time was 1-10 minutes.

[0011] (4) MALDI-MSI analysis: The sample tissue sprayed with the matrix was subjected to mass spectrometry imaging analysis to obtain the spatial distribution of metabolic components in the sample tissue. The detection mass-to-charge ratio was 100-1000 and the scanning resolution was 50 μm.

[0012] (5) Spatial transcriptome analysis: After mass spectrometry imaging, the chip was subsequently subjected to spatial transcriptome analysis.

[0013] Preferably, in step (1), the freezing sectioning temperature is -20°C ± 2°C and the time is 1-10 minutes;

[0014] Preferably, the number of spraying cycles in step (2) is 10-20 times, and the nozzle temperature is 40-60°C

[0015] Preferably, in step (3), the metal target plate includes a conductive support base having three types of square holes on the conductive support base; circular grooves of the size of the gasket are engraved on both sides of the holes, and the metal base plate is snap-fastened to the reverse side of the metal conductive support base; a spatial transcriptome chip of corresponding size can be placed in the groove, and then the gasket is placed in the groove on the chip and fixed, thereby completing the loading of the spatial transcriptome chip, and the metal target plate is made of titanium alloy.

[0016] Preferably, the size of the square cavity is 1cm×1cm×0.8mm, 0.5cm×0.5cm×0.8mm, 2.5cm×2.5cm×0.8mm, the size of the circular groove is 0.54cm×0.54cm×0.7mm, 0.7cm×0.7cm×0.7mm, 0.9cm×0.9cm×0.7mm, and the size of the metal base plate is 7.5cm×2.5cm×0.5mm.

[0017] Preferably, in step (5), the tissue permeabilization time is 10 minutes.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. The chip target plate used in this paper is detachable and can be reused; the target plate is designed with 3 specifications of spatial transcriptome chip grooves, the size of which is suitable for the BGI spatial transcriptome chip; in addition, the target plate is adapted to the thickness of the chip and will not damage the chip due to the bayonet problem; the target plate uses a titanium alloy metal target plate with uniform thickness, flat surface, and good conductivity, which can ensure the quality stability and repeatability of the mass spectrometer; finally, the target plate has a simple structure and can be mass-produced, which greatly reduces the cost of the product and is suitable for wide promotion and use;

[0020] 2. The present invention utilizes the constructed target plate to realize the spatial metabolite research of colon tissue on the spatial transcriptome chip. The detection effect has no significant difference with the effect of the traditional target plate loaded with ITO glass, and realizes the visualization characterization of metabolites in the sample tissue. The present invention realizes that the sample tissue after spatial metabolomics can still obtain qualified spatial transcriptome data, and obtains the spatial distribution information of multiple genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described below in conjunction with the accompanying drawings.

[0022] The accompanying drawings are schematic structural diagrams of this invention patent.

[0023] Figure 1 It is a schematic structural diagram of a metal target plate, the front side (A), the back side (B);

[0024] Figure 2 It is a front view of the finished target plate.

[0025] Figure 3 It is a back view of the finished target plate.

[0026] Figure 4 Mass spectrometry imaging effect diagrams of slices loaded with conductive glass on a traditional target plate (ITO - traditional target plate) and adjacent slices loaded with a spatial transcriptomics chip in the invention target plate (chip - invention target plate).

[0027] Figure 5 Representative spatial metabolism map of a spatial transcriptomics t3 tissue block.

[0028] Figure 6 Distribution map of clusters obtained by spatial transcriptomics bin50 clustering on the UMAP of the cluster; B: Distribution of each cluster of spatial transcriptomics on the spatial slice; C: ssDNA map of the spatial transcriptomics taken. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with the accompanying drawings.

[0030] Example 1

[0031] Information of the invention target plate

[0032] The material of the invented metal target plate is titanium alloy, and there are also plastic buckles. The invented target plate is composed of a conductive support base, a metal bottom plate, screws, gaskets, and a push - pull buckle. The metal bottom plate, screws, and washers are detachably fixed on the conductive support base (see Figure 1 ); The metal target plate contains three types of square grooves (the grooves formed by fixing the metal bottom plate and the bayonet on the back side of the conductive support base have models of 2.5 cm × 2.5 cm × 0.8 mm, 1 cm × 1 cm × 0.8 mm, 0.5 cm × 0.5 cm × 0.8 m respectively); The spatial transcriptomics chip can be placed in the corresponding - model grooves, and then the washer is covered on the corresponding position of the groove on the chip for fixation to achieve the loading of the spatial transcriptomics chip; The front view of the finished target plate is shown in Figure 2 , and the back view of the finished target plate is shown in Figure 3 .

[0033] Example 2

[0034] 1. Instruments and reagents

[0035] Rapiflex MALDI Tissuetyper TM TOF / TOF mass spectrometer (Bruker, Germany, equipped with SmartBeam TM 3D 355nm laser and flexControl, FlexImaging data processing workstation), ThermoCryoStar NX50 NOVPD slicer (Thermo Fisher Scientific, USA), ITO conductive glass indium tin oxide (square resistance ≤ 6Ω, South China Xiangcheng Technology Co., Ltd.), spatial transcriptome test chip (1cm×1cm, Shenzhen BGI Gene Technology Co., Ltd.), Matrix Builder matrix sprayer (Vicoto (Beijing) Technology Co., Ltd.), vacuum dryer (Shanghai Yueci Electronic Technology Co., Ltd.), SCiLS Lab 2018b, mass spectrometry imaging data analysis software (Germany GmbH).

[0036] 1,5-Diaminonaphthalene (DAN, purity > 98.0%), α-cyano-4-hydroxycinnamic acid (HCCA, purity > 98.0%), and 2,5-dihydroxybenzoic acid (DHB, purity > 98.0%) were purchased from Sigma, USA, and acetonitrile and OCT embedding medium were purchased from Beijing Xinsheng Technology Co., Ltd.

[0037] In this example, fresh mouse colon tissue (male, 20 g ± 2 g, C57BL / 6) was used, which was purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0038] 2. Sample preparation: The colon tissue was embedded with OCT embedding agent, then sliced ​​with a freezing microtome and attached to the spatial transcriptome chip and conductive glass (ITO) to obtain colon tissue slices, which were then vacuum dried at a slice temperature of -20°C ± 2°C and a slice thickness of 8 to 10 μm. The slices were vacuum dried in a vacuum dryer for 30 minutes.

[0039] 3. Matrix spraying: accurately prepare any one of the matrices of DAN, HCCA, and DHB at a concentration of 1-10 mg / mL, and the solvent is acetonitrile / water (7:3, v / v). The colon tissue after vacuum drying is sprayed with the matrix. The spraying procedure is: the number of spraying cycles is 10-20 times, the nozzle temperature is 40-60°C, the matrix solution flow rate is 0.10-0.30 mL / min, the nozzle movement spacing is 3 mm, and the nozzle movement speed is 1000-1500 mm / min;

[0040] (1) Tissue loading: The colon tissue sprayed with the matrix on the ITO is loaded onto a common metal target holder, and the position is marked on a conductive glass slide with a correction fluid, and then the glass slide area is scanned with a scanner with a scanning resolution of 1200 dpi for subsequent instrument positioning of the sample; the colon tissue sprayed with the matrix on the spatial transcriptome chip is loaded onto the invented metal target plate, and the loading time is 1-10 minutes, and the position is marked on the metal target plate with a correction fluid for subsequent instrument positioning of the sample;

[0041] (2) MALDI-MSI analysis: The colon tissue sprayed with the matrix was placed in a mass spectrometer for mass spectrometry imaging analysis. First, the imaging method was selected in FlexControl, the detection method was negative ion mode, the sample position was found, and the appropriate mass spectrometry signal was determined. Finally, the laser energy was set to 30-90%, the detection mass-to-charge ratio was 100-1000, and the method was saved. Then, a new file was created in the FlexImaging software, and the detection method was the file saved by FlexControl. Then, according to the symbol position of the correction fluid mark on the imported scanned image, the position coordinates in the two softwares were calibrated. After the calibration position was completed, the sample area was selected for imaging analysis. After the scanning was completed, the results were loaded, and the imaging data analysis was performed through SCiLS Lab 2018b.

[0042] (3) Spatial transcriptome analysis: After mass spectrometry imaging, the chip was removed from the prepared target plate and then spatial transcriptome analysis was performed. Tissue fixation, fluorescent staining and photography, tissue permeabilization, reverse transcription reaction, tissue removal, cDNA release and recovery, cDNA purification and amplification, and library construction were then performed according to the kit instructions of Qingdao Huada (Stereo-seq transcriptome reagent kit vector version).

[0043] Example 3

[0044] Spatial distribution characteristics of metabolites in colon tissue and comparison with traditional methods

[0045] Colonic tissue contains a variety of metabolites such as lipids, choline, and amino acids. This study used mass spectrometry imaging technology in negative ion mode to detect the spatial distribution of various metabolites in the cross-section of colonic tissue, such as A([MH] - ,487.051)、B([MH] - ,616.317、C([MH] -, 771.559). Among them, substance A is mainly distributed in the central annular folds, and substances B and C are mainly distributed in the muscular tissue of the periphery of the intestine. In addition, the two groups of colons showed very similar spatial distribution characteristics, and the signal intensity was relatively similar, with no significant changes. The target plate invented by these results can be applied to the imaging distribution detection of spatial transcriptome chips, and the detection effect is comparable to that of traditional target plates for detecting tissue metabolites on commonly used ITO glass.

[0046] Example 4

[0047] Effects of spatial metabolomics processing on gene quality in colon tissue on chip and comparison with traditional processing methods

[0048] (4) To study whether spatial metabolomics would affect mRNA capture, three colon tissues (named t1, t2, and t3) were attached to the same chip. The colon tissue of t3 was first subjected to spatial metabolomics and then to spatial transcriptomics. T1 and t2 were directly attached without treatment and used for subsequent idle analysis. As shown in Table 1, the number of captured genes in t3 was the lowest among the three tissue blocks, indicating that spatial metabolomics does have a certain impact on mRNA capture. However, the median gene bin50 exceeded 500, and the quality control was qualified, indicating that the impact of spatial metabolomics detection on spatial transcriptomics detection was within an acceptable range.

[0049] (5) Table 1 UMI / gene statistics after lasso splitting of three tissue blocks

[0050]

[0051] Example 5

[0052] Spatial metabolite and spatial transcriptome analysis on the same transcriptome chip

[0053] The prepared target plate was used to detect and spatially distribute multiple substances on the cross-section of colon tissue. Multiple molecular weight substances were detected on the t3 tissue block, showing different spatial distribution characteristics, such as m / z 267.023, m / z 487.130, m / z 616.371, m / z 687.431 and m / z 766.410. This result proves that the target plate is suitable for the detection of spatial metabolites on colon tissue on the spatial transcriptome chip ( Figure 5 ).

[0054] Based on bin 50 data, cluster analysis was performed using Seurat, and cells with a gene count of <200 or >3000 or a mitochondrial gene ratio of >5% were filtered out. Resolution was set to 1, and a total of 11 clusters were obtained ( Figure 6). This result proves that the colon tissue after spatial metabolomics processing still retains a large amount of RNA and has a clear spatial distribution

[0055] In summary, the present invention discloses a method for realizing MALDI mass spectrometry imaging spatial metabolomics and spatial transcriptomics detection on the same tissue section based on a novel target plate, develops a durable target plate suitable for MALDI-TOF mass spectrometry that can realize spatial metabolite detection of biological tissues on a spatial transcriptome chip, and applies it to the detection of metabolites in mouse colon tissue. Subsequently, the tissue sections on the spatial transcriptome chip can continue to perform in situ detection of genes in spatial transcriptomics, thereby realizing the combined detection of spatial metabolomics and spatial transcriptomics on the same tissue section. The target plate of the invention consists of a conductive support seat, a metal base plate, a gasket, and a push-pull buckle. The metal base plate and the gasket are detachably fixed on the conductive support seat; the metal target plate contains three types of square grooves (the metal base plate and the buckle are fixed on the back of the conductive support seat, and the groove types formed are 2.5cm×2.5cm×0.8mm, 1cm×1cm×0.8mm, and 0.5cm×0.5cm×0.8m respectively); the spatial transcriptome chip can be placed in the groove of the corresponding model, and then the gasket is covered on the corresponding position of the groove on the chip, and the screws are tightened to realize the loading of the spatial transcriptome chip.

[0056] The spatial metabolite detection includes the following steps: first, the colon tissue is embedded with an embedding agent, then sliced ​​by a freezing microtome and attached to a conductive glass (ITO) and a spatial transcriptome chip respectively, and dried; then the colon tissue slices are sprayed with a matrix; then the colon tissue sprayed with the matrix is ​​subjected to MALDI-TOF mass spectrometry imaging analysis to obtain the spatial distribution of metabolites in the colon tissue; spraying the matrix; then the imaged colon tissue is subjected to spatial transcriptome analysis to obtain the spatial distribution characteristics of colon genes. The present invention prepares a durable MALDI-TOF target plate capable of loading biological tissue on a spatial transcriptome chip. In a negative ion detection mode, a MALDI mass spectrometry imaging analysis method is used to establish a high-sensitivity and visualized mass spectrometry imaging method for metabolites in colon tissue on a spatial transcriptome chip. In addition, good spatial transcriptome data of colon tissue on the same chip are obtained. The target plate overcomes the problems that ordinary target plates cannot fix chips of this model, the thickness of the card slot design is not suitable for the chip thickness, and the card slot design is difficult to meet the requirement that the chip surface coating cannot be damaged. The target plate has high sensitivity, obtains good spatial transcriptome and spatial metabolome data on the same tissue slice, and provides technical support for the joint analysis of spatial metabolomics and spatial transcriptomics.

Claims

1. A method for MALDI mass spectrometry imaging and spatial transcriptomics detection on the same tissue section, characterized in that: The following steps are involved: Sample preparation: embed the sample tissue with an embedding agent, then slice it with a freezing microtome, stick it on the spatial transcriptome chip and conductive glass, obtain tissue slices, and perform vacuum drying; Matrix spraying: spraying the sample tissue after vacuum drying with a matrix, wherein the matrix is ​​any one of DAN, HCCA and DHB, and obtaining tissue sections sprayed with the matrix; Tissue loading: The colon tissue sprayed with matrix on the conductive glass was loaded onto a common metal target holder; the colon tissue sprayed with matrix on the spatial transcriptome chip was loaded onto the corresponding groove of the metal target plate, and the loading time was 60±20 s; MALDI-MSI analysis: The sample tissue sprayed with the matrix was subjected to mass spectrometry imaging analysis to obtain the spatial distribution of metabolic components in the sample tissue. The laser energy was set to 30-90%, the detection mass-to-charge ratio was 100-1000, and the scanning resolution was 50 μm. Spatial transcriptome analysis: After mass spectrometry imaging, the chip was subsequently subjected to spatial transcriptome analysis.

2. The method according to claim 1, characterized in that: In step (1), the freezing section slicing temperature is -20°C ± 2°C and the time is 1 to 10 minutes.

3. The method according to claim 1, characterized in that In step (2), the number of spraying cycles is 10-20 times, and the nozzle temperature is 40-60°C.

4. The method according to claim 1, characterized in that: In step (3), the metal target plate includes a conductive support base, which has three types of square holes; circular grooves of the size of gaskets are engraved on both sides of the holes, and the metal base plate is snap-fastened to the reverse side of the metal conductive support base, and the metal base plate and the conductive support base are conductively connected; a spatial transcriptome chip of corresponding size can be placed in the groove, and then the gasket is placed in the groove on the chip and fixed, thereby completing the loading of the spatial transcriptome chip, and the metal target plate is made of titanium alloy.

5. The method according to claim 1, characterized in that The dimensions of the square holes are 1cm×1cm×0.8mm, 0.5cm×0.5cm×0.8mm, 2.5cm×2.5cm×0.8mm, the dimensions of the circular grooves are 0.54cm×0.54cm×0.7mm, 0.7cm×0.7cm×0.7mm, 0.9cm×0.9cm×0.7mm, and the dimensions of the metal base plate are 7.5cm×2.5cm×0.5mm.

6. The method according to claim 1, characterized in that The time for tissue permeabilization was 10 minutes.

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