Synergistic matrix for mass spectrum imaging and preparation method and application thereof

Abstract

The invention belongs to the technical field of mass spectrum imaging, and particularly relates to a synergistic matrix for mass spectrum imaging and a preparation method and application thereof. The invention provides a proton sponge type MALDI-MSI synergistic matrix formed on the basis of 2-hydrazinoquinoline and alpha-cyano-4-hydroxycinnamic acid, aiming at the problems of non-uniform crystallization, strong background interference, different ionization efficiency and complex matrix interference of a biological sample in the traditional MALDI matrix. The ionization efficiency of various metabolites in mass spectrometry can be remarkably improved by means of the uniform crystal morphology and the auxiliary target analyte desorption / ionization capacity. In the application of mass spectrum imaging, the synergistic matrix not only greatly improves the detection signal intensity, but also improves the spatial reproducibility, and provides an effective solution for synchronous high-sensitivity mass spectrum imaging of multiple types of small molecule metabolites in complex biological samples.

Description

A synergistic matrix for mass spectrometry imaging, its preparation method and application Technical Field

[0001] This invention belongs to the field of mass spectrometry imaging technology, specifically relating to a synergistic matrix for mass spectrometry imaging, its preparation method, and its application. Background Technology

[0002] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) is a technique that combines mass spectrometry analysis with spatial imaging, enabling the in-situ acquisition of two-dimensional distribution information of molecules such as metabolites and lipids in tissue sections. However, MALDI-MSI faces significant challenges when imaging small molecule metabolites (such as biomarkers of oxidative stress): on the one hand, small molecule metabolites generally have low ionization efficiency, resulting in weak signals and insufficient detection sensitivity; on the other hand, endogenous compounds present in the tissue matrix and the matrix itself easily generate high-intensity background interference, severely affecting the specificity and accuracy of the target metabolite signal.

[0003] Monitoring reactive carbonyl compounds (RCSs) and their mediated lipid peroxidation is crucial for elucidating the molecular mechanisms of oxidative stress. However, current MALDI-MSI-based studies still face significant challenges in achieving simultaneous spatial imaging of RCSs and their related metabolites, such as low-molecular-weight metabolites and lipids. This is mainly due to the significant differences in ionization efficiency among different types of metabolites under MALDI conditions, with carbonyl metabolites such as RCSs being difficult to detect effectively due to their challenging ionization process.

[0004] The matrix plays a crucial role in MALDI-MSI. Traditional MALDI matrices, such as α-cyano-4-hydroxycinnamic acid (CHCA), N1,N4-dibenzylphenyl-1,4-diamine (DBDA), 2,5-dihydroxybenzoic acid (DHB), and sinapic acid (SA), typically have limitations, including inhomogeneous crystallization with the analyte and the generation of abundant matrix background signals. Furthermore, there are issues with the ionization capabilities of different compounds and interference from complex matrices in biological samples. These limitations restrict the comprehensive application of MALDI-MSI in bioimaging. Summary of the Invention

[0005] The purpose of this invention is to provide a MALDI-MSI co-matrix that can significantly improve the sensitivity and imaging quality for detecting reducing metabolites (such as glutathione, GSH), reactive carbonyl substances (RCSs), and lipids in biological tissues. Based on a "proton sponge" type co-matrix formed from 2-hydrazinoquinoline (2-HQ) and α-cyano-4-hydroxycinnamic acid (CHCA), this co-matrix, with its uniform crystal morphology and ability to assist in the desorption / ionization of target analytes, can significantly improve the ionization efficiency of various metabolites in mass spectrometry analysis. In imaging applications, this system not only greatly improves the detection signal intensity of RCSs but also enhances spatial reproducibility, providing an effective solution for simultaneous high-sensitivity mass spectrometry imaging of multiple classes of small molecule metabolites in complex biological samples.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A first aspect of the present invention provides a co-matrix for mass spectrometry imaging, comprising 2-hydrazinoquinoline (2-HQ) and α-cyano-4-hydroxycinnamic acid (CHCA); wherein the 2-HQ and CHCA are co-dissolved in an acetonitrile / water solution containing trifluoroacetic acid, wherein the volume ratio of acetonitrile to water is 6:4, and the volume fraction of the trifluoroacetic acid is 0.1%.

[0008] Furthermore, as a more advanced technical solution of the present invention, the mass ratio of 2-hydrazinoquinoline to α-cyano-4-hydroxycinnamic acid is (0.1~10):10.

[0009] A second aspect of the present invention provides a method for preparing a co-matrix for mass spectrometry imaging, comprising the following steps:

[0010] Step 1: Prepare stock solutions of 2-hydrazinoquinoline and α-cyano-4-hydroxycinnamic acid, respectively;

[0011] Step 2: Mix the 2-hydrazinoquinoline stock solution and the α-cyano-4-hydroxycinnamic acid stock solution in a certain proportion to prepare the co-matrix working solution.

[0012] Furthermore, as a more advanced technical solution of the present invention: the concentration of the 2-hydrazinoquinoline stock solution is 0.1~10 mg / mL, and the concentration of the α-cyano-4-hydroxycinnamic acid stock solution is 10 mg / mL.

[0013] A third aspect of the invention provides the application of the co-matrix in monitoring changes in multiple metabolites during oxidative stress in MALDI-MSI.

[0014] Furthermore, as a more advanced technical solution of the present invention, the metabolites during the oxidative stress process include spermine, spermidine, 3-aminopropionaldehyde (AMP), 3-acetylaminopropionaldehyde (ACP), acrolein (ACR), glutathione (GSH), arachidonic acid (AA), N-acetyl-S-(3-hydroxypropyl)cysteine ​​(3-HPMA), and phosphatidylcholine (PCs).

[0015] Furthermore, as a more advanced technical solution of the present invention: the application of the co-matrix in monitoring changes in multiple metabolites during oxidative stress in MALDI-MSI includes the following steps:

[0016] Step 1: Sacrifice the experimental rats, then remove the spinal cord tissue and immediately place it in liquid nitrogen for cryopreservation;

[0017] Step 2: At -20°C, use a Leica CM1900 cryostat to cut the frozen rat spinal cord tissue from Step 1 into 12-micron-thick tissue sections.

[0018] Step 3: Place the tissue sections from Step 2 onto an ITO conductive glass slide and dry them in a vacuum for 30 minutes;

[0019] Step 4: Use a spray gun to uniformly spray the co-matrix solution provided in the first aspect of the present invention onto the surface of the tissue section to form a matrix layer;

[0020] Step 5: Using a matrix-assisted laser desorption / ionization mass spectrometry imaging system, perform mass spectrometry imaging analysis on tissue sections coated with a matrix layer to obtain the mass spectrometry signals of metabolites and their spatial distribution images;

[0021] Step 6: After generating and exporting a list of single isotope peaks from the raw mass spectrometry imaging data, the measured mass-to-charge ratio data are compared with two metabolomics databases—METLIN37 and LIPID MAPS—to identify metabolites. Ion images of the identified metabolites are then reconstructed using SCiLS Lab 2022b Pro software.

[0022] Furthermore, as a more advanced technical solution of the present invention: in step five, mass spectrometry imaging uses a positive ion reflector mode for data acquisition.

[0023] The beneficial effects of this invention are:

[0024] 1. Compared with using CHCA or 2-HQ alone, the co-matrix provided by this invention achieves an order-of-magnitude improvement in the detection signal intensity of a variety of oxidative stress-related metabolites. Studies have shown that 2-HQ and CHCA in the co-matrix form a "mass spectrometry sponge" that is conducive to the ionization of target analytes in mass spectrometry.

[0025] 2. The co-matrix forms extremely uniform and fine co-crystals on the tissue surface, effectively avoiding the "hot spot" effect. This results in MALDI mass spectrometry images with lower background noise, higher spatial resolution, and clearer tissue structure correspondence, enabling a more realistic and detailed reflection of the in-situ spatial distribution of metabolites.

[0026] 3. The co-matrix preparation method provided by this invention is simple and reproducible, and is suitable for high-sensitivity imaging of multiple metabolites in biological oxidative stress models, providing a powerful tool for the study of oxidative stress mechanisms and the spatial localization of disease biomarkers. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art are briefly introduced below; obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0028] Figure 1 shows the average MALDI-MSI spectra obtained from rat spinal cord tissue sections with different ratios of co-matrix in Example 1; wherein, the concentration of 2-HQ was set to 0.1 mg / mL, 1.0 mg / mL, 5.0 mg / mL and 10 mg / mL, respectively, and the concentration of CHCA was kept constant at 10 mg / mL;

[0029] Figure 2 shows the MALDI MS spectra of three carbonyl standards using 2-HQ and co-matrix as matrices in Example 2; where, top: 2-HQ (aqueous solution) / standard; middle: 2-HQ (rat spinal cord homogenate) / standard; bottom: co-matrix (rat spinal cord homogenate) / standard;

[0030] Figure 3 shows the ion intensity diagrams obtained by MALDI mass spectrometry detection of 15 compounds using different matrices (co-matrix, 2-HQ, CHCA, DBDA, DHB) in Example 3 and Comparative Example 1; ns: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; Sample size n=3;

[0031] Figure 4 shows the metabolite-driven spatial segmentation (SM), optical images of homogenized spinal cord simulated slices, and scanning electron microscope (SEM) images of the coated co-matrix, 2-HQ, and CHCA in Example 4; wherein the scale bar of the spatial segmentation and optical images is 1 mm; and the scale bar of the scanning electron microscope images is 5 μm.

[0032] Figure 5 shows the mass spectrometry analysis of the co-matrix in Example 2; wherein, A in Figure 5 is the MALDI mass spectrum of 2-HQ, CHCA and co-matrix; B in Figure 5 is the secondary mass spectrum (MS / MS) of the ion at a mass-to-charge ratio (m / z) of 349.0; C in Figure 5 is a schematic diagram of the formation of the proton-bound 2-HQ (B) and CHCA (AH) ion pair.

[0033] Figure 6 shows the average mass spectra of low molecular weight metabolites (m / z < 400) and lipids (450 < m / z < 900) using co-matrix, CHCA, and 2-HQ as matrices.

[0034] Figure 7 shows the spatial distribution of selected metabolites in rat spinal cord tissue sections using co-matrix, CHCA and 2-HQ matrices. The scale bar is 1 mm.

[0035] Figure 8 shows the principal component analysis (PCA) scatter plot of the MALDI MS spectra of rat spinal cord homogenates obtained using co-matrix, 2-HQ, CHCA, DBDA, and DHB. The figure analyzes 25 metabolites in the spinal cord homogenate, and each sample was tested three times. The ellipse represents the 85% confidence interval of the data distribution area for each group. Detailed Implementation

[0036] To make the objectives, technical effects, and technical solutions of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention. Based on the embodiments disclosed in the present invention, other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0037] The matrix-assisted matrix desorption / ionization time-of-flight mass spectrometer used in the following examples was an ultrafleXtreme TOF / TOF (Bruker Daltonics, Bremen, Germany), employing a 355 nm pulsed Nd:YAG laser. All mass spectra were acquired in positive ion reflectance mode within a mass-to-charge ratio (m / z) range of 0-1000: accelerating voltage, 25.00 kV; delayed extraction voltage, 23.20 kV; delayed extraction time, 400 ns; frequency, 1000 Hz; spatial resolution set to 60 μm. Data acquisition was performed using flexImaging 4.1 software, and mass spectrometry imaging data were processed and visualized using SCiLS Lab 2022bPro software. Following MALDI-MSI analysis, the slides were stained with hematoxylin and eosin (H&E).

[0038] Animal experiments: Female Sprague-Dawley rats (weighing 180-200 grams) were purchased from the Experimental Animal Center of Jilin University. A spinal cord injury model was established using a combination of a drop test and a spinal cord impact device. The procedure was as follows: After anesthetizing the rats, the T10 segment of the spinal cord was exposed, and an impact rod (weighing 40 grams, dropped from a height of 6 cm, and with a diameter of 2.5 mm) was used to impact the dorsal side of the spinal cord. Postoperatively, penicillin was injected daily to prevent infection, and bladder massage was performed twice daily to assist urination. The rats were sacrificed at predetermined time points, and the spinal cord tissue was immediately frozen in liquid nitrogen. All animal procedures were approved by the relevant departments of Jilin University.

[0039] Example 1: Optimization of the ratio of 2-HQ to CHCA solution in a co-matrix

[0040] (1) Prepare 2-HQ solutions with mass concentrations of 0.1, 1, 5 and 10 mg / mL respectively, wherein the solvent is an acetonitrile / water solution containing trifluoroacetic acid, wherein the volume ratio of acetonitrile to water is 6:4, and the volume fraction of trifluoroacetic acid is 0.1%;

[0041] (2) Prepare a CHCA stock solution with a mass concentration of 10 mg / mL, wherein the solvent is an acetonitrile / water solution containing trifluoroacetic acid, wherein the volume ratio of acetonitrile to water is 6:4, and the volume fraction of trifluoroacetic acid is 0.1%;

[0042] (3) Take 3 mL of 2-HQ solution from step (1) and 3 mL of CHCA stock solution from step (2) and mix them to prepare co-matrix working solution;

[0043] (4) Preparation of homogenized spinal cord simulated tissue sections: healthy rat spinal cord tissue was weighed, ground and pulverized, and transferred to centrifuge tubes; homogenization was performed at a ratio of 5 μL of 1% sodium dodecyl sulfate (SDS) solution per milligram of tissue to prepare rat spinal cord homogenate; 100 μL of homogenate was injected into a 1 mL disposable syringe (with the end cut off), frozen in liquid nitrogen for 30 seconds, and then extruded into a columnar frozen homogenate. The homogenate was immediately transferred at a uniform speed to an ITO conductive glass slide to prepare thawed and attached sections of uniform thickness. The sections were vacuum dried for 30 minutes.

[0044] (5) Spraying the matrix: Take the homogeneous spinal cord simulated tissue section from step (4) and send it into the matrix sprayer (Model 200, Badger Air-Brush, Washington, USA). Take 5 mL of the co-matrix working solution from step (3) for matrix spraying. The specific operation steps are as follows: use a spray gun to spray the matrix, set the compressed air pressure to 10 psi, the solvent flow rate to 1 mL / min, the nozzle temperature to 60℃, and the matrix solution uniformly covers the surface of the section by 20 reciprocating sprays.

[0045] (6) A matrix-assisted laser desorption / ionization mass spectrometry imaging system was used to perform mass spectrometry imaging analysis on tissue sections coated with a matrix layer to obtain the mass spectrometry signals and spatial distribution images of metabolites. After generating and exporting a list of single isotope peaks from the raw mass spectrometry imaging data, the identified metabolites were reconstructed using SCiLS Lab 2022b Pro software. In the data preprocessing stage, the “TopHat” algorithm was first used for baseline subtraction, followed by root mean square normalization, and then the obtained average spectra were compared. The average ion intensity of the low molecular weight metabolite region (0 < m / z < 450) and the lipid region (500 < m / z < 900) was analyzed.

[0046] Example 2: MALDI-MS analysis based on different matrices: Mass spectra of the detection of three carbonyl compound standards in rat spinal cord homogenate or aqueous matrix under different matrices.

[0047] The three carbonyl compounds include: acrolein (ACR), benzaldehyde (BA), and acetophenone (ACT).

[0048] (1) Prepare 2-HQ solution and CHCA solution with a mass concentration of 10 mg / mL respectively, wherein the solvent is an acetonitrile / water solution containing trifluoroacetic acid, wherein the volume ratio of acetonitrile to water is 6:4, and the volume fraction of trifluoroacetic acid is 0.1%;

[0049] (2) Prepare a mixed aqueous solution of ACR, BA, and ACT with a mass concentration of 1 mg / mL;

[0050] (3) Take 1 mL of rat spinal cord homogenate from step (4) of Example 1, add 1 mg of ACR, BA and ACT respectively, and prepare spiked spinal cord homogenate solution;

[0051] (4) Take 1 μL of 2-HQ solution from step (1) and 1 μL of aqueous solution of the three carbonyl standards from step (2) and mix them evenly on a MALDI target plate.

[0052] (5) Take 1 μL of 2-HQ solution from step (1) and 1 μL of spiked spinal cord homogenate from step (3) and mix them evenly on a MALDI target plate.

[0053] (6) Take 1 μL of the co-matrix working solution with a mass ratio of 2-HQ:CHCA=1:1 in step (3) of Example 1 and 1 μL of the spiked spinal cord homogenate in step (3), and mix them evenly on a MALDI target plate.

[0054] (7) Perform MALDI testing on the spotting in steps (4) to (6) and select positive ion reflection mode.

[0055] Example 3: Investigating the ionic intensity of 15 metabolites detected by MALDI mass spectrometry using a co-matrix.

[0056] The 15 compounds in this embodiment include: acrolein (ACR), acetone, benzaldehyde (BA), trans-9-octadecenoic alcohol, spermidine, spermine, linoleic acid, trans-11-eicosenoic acid, arachidonic acid (AA), myristic acid, nervonic acid, arginine, serine, threonine, and glutathione (GSH).

[0057] (1) Prepare aqueous solutions of 15 metabolite standard samples: 1 mg / mL;

[0058] (2) Take 1 μL of sample solution and 1 μL of the optimal ratio of co-matrix working solution in step (3) of Example 1 and add them to the same point on the Anchorchip target plate, and mix them directly with a pipette tip;

[0059] (3) The target plate is sent into the MALDI mass spectrometer and data acquisition is performed using the positive ion reflection mode.

[0060] Example 4: Investigating the co-crystallization of different matrices with rat spinal cord homogenate

[0061] (1) Take the homogeneous spinal cord simulated tissue slices from step (4) of Example 1 and send them into a matrix sprayer (Model 200, Badger Air-Brush, Washington, USA). Take 5 mL of 1 mg / mL 2-HQ solution from step (1) of Example 1 and spray it onto the matrix.

[0062] (2) Take the homogeneous spinal cord simulated tissue slices from step (4) of Example 1 and send them into a matrix sprayer (Model 200, Badger Air-Brush, Washington, USA). Take 5 mL of the 10 mg / mL CHCA solution from step (2) of Example 1 and spray it onto the matrix.

[0063] (3) Take a slice of homogeneous spinal cord simulated tissue after co-matrix spraying in step (5) of Example 1;

[0064] (4) Perform imaging tests and data analysis on the tissue sections from steps (1) to (3) according to step (6) of Example 1, and perform partition (SM) data processing.

[0065] Example 5: Investigating the mass spectrometry imaging effect of co-matrix

[0066] (1) Tissue section preparation: The frozen rat spinal cord tissue was cut into 12 μm thick sections at -20℃ using a Leica CM1900 cryostat; the continuous tissue sections were immediately thawed and attached to the conductive surface of ITO conductive glass slides;

[0067] (2) Spray co-matrix onto the dried tissue sections. The specific operation steps are as follows: inject 5 mL of co-matrix into the spray gun for matrix spraying. Set the compressed air pressure to 10 psi, the solvent flow rate to 1 mL / min, and the nozzle temperature to 60 ℃. The matrix solution is sprayed 20 times to evenly cover the surface of the section.

[0068] (3) The tissue sections coated with the matrix were subjected to imaging tests in a mass spectrometer. The sample was loaded into the mass spectrometer, the positive ion reflection mode was selected, the laser energy was 70%, the laser wavelength was 355nm, the laser spot diameter was selected as small, and the instrument imaging resolution was selected as 60μm.

[0069] (4) Data acquisition was completed using flexImaging 4.1 software. Mass spectrometry imaging data were processed and visualized using SCiLS Lab2022b Pro software. After MALDI-MSI analysis, the slides were stained with hematoxylin and eosin (H&E).

[0070] Example 6

[0071] (1) Prepare matrix solutions of 2-HQ, CHCA, DBDA, and DHB with a concentration of 1 mg / mL respectively:

[0072] (2) Take 1 μL of matrix solution from step (1) and 1 μL of rat spinal cord homogenate from step (4) of Example 1 and add them to the same point on the Anchorchip target plate, and mix them directly with a pipette tip;

[0073] (3) Take 1 μL of the optimal ratio of co-matrix solution in step (3) of Example 1 and 1 μL of rat spinal cord homogenate in step (4) of Example 1 and add them sequentially to the same point on the Anchorchip target plate, and mix them directly using a pipette tip;

[0074] (4) The target plate is sent into the MALDI mass spectrometer and data acquisition is performed using the positive ion reflection mode.

[0075] (5) The measured mass-to-charge ratio data were compared with two metabolomics databases—METLIN37 and LIPID MAPS—to analyze 25 metabolite peaks in rat spinal cord homogenate samples. The first three principal components extracted by PCA were used as input features for a Python classification model, and the model was trained using linear discriminant analysis (LDA), support vector machine (SVM), and k-nearest neighbor (KNN) algorithms. The performance of five matrices (co-matrix, 2-HQ, CHCA, DBDA, and DHB) was systematically evaluated.

[0076] 25 metabolites: glycine (Gly), alanine (Ala), proline (Pro), purine, nicotinic acid, lysine (Lie), ornithine, aspartic acid (Asp), glutamine (Gln), methionine (Met), histidine (His), carnitine, phenylalanine (Phe), tyrosine (Tyr), sulamine, linoleic acid, arachidonic acid, nervonic acid, diacylglycerol (DG 30:1), phosphatidic acid (PA40:6), phosphatidylglycerol (PG 36:3), phosphatidylcholine (PC 36:5), sphingomyelin (SM d40:1), phosphatidylethanolamine (PE 42:4), and phosphatidylcholine (PC 40:10).

[0077] Comparative Example 1: Investigating the ionic intensity of 15 metabolites detected by MALDI mass spectrometry using 2-HQ, CHCA, DBDA, and DHB matrices.

[0078] This comparative example provides MALDI mass spectrometry analysis of 15 metabolites using different matrices. The operation method is basically the same as that in Example 3. The main difference is that the matrices used in step (2) are 2-HQ, CHCA, DBDA and DHB, with a concentration of 10 mg / mL.

[0079] Comparative Example 2: The mass spectrometry imaging performance of 2-HQ and CHCA matrices was examined respectively.

[0080] This comparative example provides mass spectrometry imaging results for 2-HQ and CHCA matrices, respectively. The operation method is basically the same as that in Example 5, with the main difference being that 2-HQ and CHCA matrices are used instead of co-matrix, respectively.

[0081] Figure 1 shows the average MALDI-MSI spectra obtained from rat spinal cord tissue sections with different ratios of co-matrix in Example 1. As can be seen from Figure 1, when the 2-HQ concentration is 1 mg / mL and the CHCA concentration is 10 mg / mL, the absolute ionic intensities corresponding to low-molecular-weight metabolites (m / z < 400) and lipids (450 < m / z < 900) are higher than those at other concentrations. Therefore, the optimal concentration of 2-HQ was determined to be 1 mg / mL.

[0082] Figure 2 shows the MALDI-MS spectra of three carbonyl standards using 2-HQ and co-matrix as matrices in Example 2. As shown in the middle spectrum of Figure 2, compared with the detection results in aqueous phase (top spectrum of Figure 2), the peak intensity and signal-to-noise ratio of [(2-HQ+ACR)+H]⁺ and [(2-HQ+BA)+H]⁺ in rat spinal cord homogenate were both reduced. Furthermore, when 2-HQ was used as the MALDI-MS matrix, the ion peak of [(2-HQ+ACT)+H]⁺ was not observed in rat spinal cord homogenate. The synergistic matrix successfully achieved on-target derivatization and detection of the three carbonyl standards in rat spinal cord homogenate. As shown in the bottom spectrum of Figure 2, compared with using the 2-HQ matrix alone, the peak intensity and signal-to-noise ratio of the three 2-HQ adduct carbonyl compound ions were significantly improved after using the synergistic matrix.

[0083] Figure 3 shows the ion intensity maps obtained by MALDI mass spectrometry detection of 15 metabolite standards using different matrices (co-matrix, 2-HQ, CHCA, DBDA, DHB) in Example 3 and Comparative Example 1. As shown in Figure 3, the synergistic matrix showed significantly higher signal intensities than any other matrix for eleven metabolites, including three carbonyl standards, four fatty acids, spermine, spermine, transoleol, and arachidonic acid (AA). The detection intensity of the synergistic matrix was also superior to other matrices for three amino acids (arginine, serine, and threonine) and glutathione (GSH).

[0084] Figure 4 shows the metabolite-driven spatial segmentation (SM), optical images of homogenized spinal cord simulated slices, and scanning electron microscopy (SEM) images of the co-matrix, 2-HQ, and CHCA sprayed in Example 4. The representative metabolite-driven segmentation patterns shown in Figure 4 visually reflect point-to-point reproducibility: the segmentation pattern of the synergistic matrix presents a single, uniform color patch, indicating that the metabolite fingerprint has high reproducibility and a consistent ion intensity distribution; while when 2-HQ or CHCA is used alone, obvious color differentiation reveals the spatial heterogeneity present in the MALDI-MSI (caused by the "sweet spot effect"). We believe that the improved reproducibility of the synergistic matrix stems from its uniform crystallization characteristics—SEM observation confirms that the sprayed synergistic matrix forms uniform, fine co-crystals, while 2-HQ and CHCA produce irregular, heterogeneous crystal structures on the silicon substrate.

[0085] Figure 5 shows the presence of the "proton sponge" confirmed by mass spectrometry analysis of the co-matrix. As can be observed in Figure 5, a characteristic peak of [(2-HQ+CHCA)+H]⁺ appears at m / z 349.0 in the mass spectrum of the co-matrix. Further identification of this ion structure by LIFT tandem mass spectrometry analysis: As shown in Figure B, the precursor ion (m / z 349.0) generates a significant fragment ion at m / z 159.2 through a neutral loss of 189.8 Da (CHCA molecular weight), which was identified as [2-HQ]⁺. Previous studies have reported that the formation of acid-base ion pairs (B⁺···H···A⁻) in different co-matrix systems can significantly improve the sensitivity and repeatability of MSI detection. As shown in Figure C, we observed that the conjugate organic base B (2-HQ) and the organic acid HA (CHCA) form a zwitterion pair. This result further demonstrates that this zwitterion pair not only promotes the formation of homogeneous co-crystallization of analytes but also simultaneously assists in the ionization process of acidic / basic analytes. Furthermore, the specific derivatization reaction of 2-HQ on carbonyl compounds enables comprehensive MSI imaging analysis of metabolites in real organic tissue sections.

[0086] Figure 6 shows the average mass spectra of low molecular weight metabolites (m / z < 400) and lipids (450 < m / z < 900) using co-matrix, CHCA, and 2-HQ as matrices. As shown in Figure 6, when using the co-matrix, the absolute ionic intensities and peak numbers of low molecular weight metabolites (m / z < 400) and lipids (450 < m / z < 900) were significantly higher than those using CHCA or 2-HQ compared to CHCA and 2-HQ alone. The detection intensities of various metabolites increased by 1-3963 times and 4-5681 times compared to those using CHCA and 2-HQ, respectively. Furthermore, the high sensitivity of the co-matrix allowed for clear visualization of the distribution of 12 selected metabolites in the gray and white matter of the rat spinal cord. As one of the most abundant classes of phospholipids and a major building block of biomembranes, the differentiated distribution patterns of phosphatidylcholine (PCs) are associated with structural and functional differences in specific regions of the spinal cord.

[0087] Figure 7 shows the spatial distribution of selected metabolites in rat spinal cord tissue sections using co-matrix, CHCA, and 2-HQ matrices. All images were normalized using SCiLS Lab 2022b Pro software. Figure 7 shows the protonated ions of PCs (e.g., [PC(36:4)+H] at m / z 782.6). + [PC(38:5)+H] of m / z 808.6 + and [PC(40:10)+H] at m / z 826.5. + In gray matter, lysophosphatidylglycerol (LPG) (18:1) was enriched, while PC (P-32:1) did not show region-specific distribution. Changes in lysophosphatidylglycerol levels due to lipid hydrolysis following spinal cord compression have also been reported. Dysglycerol (DGs), as endogenous lipid intermediates, also exhibited structural differentiation—DG (34:4) predominantly distributed in white matter revealed region-specific lipid metabolism. Furthermore, low-molecular-weight metabolites associated with oxidative stress (including spermidine, spermine, 3-HPMA, AA, and GSH) preferentially distributed in white matter, while acrolein derivatives (m / z 198.3 of [(ACR+2-HQ)+H]) were more readily distributed. + The distribution of these metabolites is non-uniform. These results indicate that the synergistic matrix is ​​an effective tool for detecting and imaging metabolites associated with reactive carbonyl compounds, laying a methodological foundation for in-depth research on their dynamic changes under oxidative stress.

[0088] Figure 8 shows the PCA scatter plot of MALDI-MS spectra obtained using co-matrix, 2-HQ, CHCA, DBDA, and DHB. Figure 8 illustrates the distribution of samples stained according to their corresponding matrices in the PC1 / PC2 score plot. Data points from the co-matrix are concentrated on the right side of the PCA plot, while 2-HQ, DBDA, and DHB cluster on the left side. Furthermore, 2-HQ / CHCA and CHCA are clearly distinguishable along the PC2 axis. This indicates that the co-matrix exhibits superior MALDI-MS detection performance and higher sensitivity for low molecular weight metabolites and lipids.

[0089] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still make modifications or equivalent substitutions to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are within the protection scope of the claims of the present invention pending approval.

Claims

1. A synergistic matrix for mass spectrometry imaging, characterized in that, It is composed of 2-hydrazinoquinoline and α-cyano-4-hydroxycinnamic acid; the 2-hydrazinoquinoline and α-cyano-4-hydroxycinnamic acid are dissolved together in an acetonitrile / water solution containing trifluoroacetic acid, wherein the volume ratio of acetonitrile to water is 6:4, and the volume fraction of trifluoroacetic acid is 0.1%.

2. The synergistic matrix for mass spectrometry imaging according to claim 1, characterized in that, The mass ratio of 2-hydrazinoquinoline to α-cyano-4-hydroxycinnamic acid is (0.1~10):

10.

3. A method for preparing a synergistic matrix for mass spectrometry imaging according to any one of claims 1 to 2, characterized in that, Includes the following steps: Step 1: Prepare stock solutions of 2-hydrazinoquinoline and α-cyano-4-hydroxycinnamic acid respectively; Step 2: Mix the 2-hydrazinoquinoline stock solution and the α-cyano-4-hydroxycinnamic acid stock solution in a certain proportion to obtain the synergistic matrix working solution.

4. The preparation method according to claim 3, characterized in that, In step one, the concentration of the 2-hydrazinoquinoline stock solution is 0.1~10 mg / mL, and the concentration of the α-cyano-4-hydroxycinnamic acid stock solution is 10 mg / mL.

5. The application of the synergistic matrix according to any one of claims 1 to 2 in mass spectrometry imaging of metabolite changes during oxidative stress.

6. The application of the synergistic matrix according to claim 5 in mass spectrometry imaging of metabolite changes during oxidative stress, characterized in that, The metabolites produced during the oxidative stress process include spermine, spermidine, 3-aminopropionaldehyde, 3-acetylaminopropionaldehyde, acrolein, glutathione, arachidonic acid, N-acetyl-S-(3-hydroxypropyl)cysteine, and phosphatidylcholine.

7. The application of the synergistic matrix according to claim 5 in mass spectrometry imaging of metabolite changes during oxidative stress, characterized in that, Includes the following steps: Step 1: Sacrifice the experimental rats, then remove the spinal cord tissue and immediately freeze it in liquid nitrogen; Step 2: At -20°C, use a Leica CM1900 cryostat to cut the frozen rat spinal cord tissue from Step 1 into 12-micron-thick tissue slices; Step 3: Place the tissue slices from Step 2 on an ITO conductive glass slide and dry them in a vacuum for 30 minutes; Step 4: Use a spray gun to uniformly spray the synergistic matrix solution described in any one of claims 1-2 onto the surface of the tissue slices to form a matrix layer; Step 5: Use a matrix-assisted laser desorption / ionization mass spectrometry (MADS) imaging system to perform mass spectrometry imaging analysis on the tissue slices coated with the matrix layer to obtain the mass spectrometry signals and spatial distribution images of the metabolites; Step 6: After generating and exporting a list of single isotope peaks from the raw mass spectrometry imaging data, compare the measured mass-to-charge ratio data with two metabolomics databases—METLIN37 and LIPID MAPS—to identify the metabolites, and use SCiLS Lab 2022b Pro software to reconstruct the ion images of the identified metabolites.

8. The application of the synergistic matrix according to claim 7 in mass spectrometry imaging of metabolite changes during oxidative stress, characterized in that, In step five, mass spectrometry imaging uses the positive ion reflector mode for data acquisition.

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