A 9-AA matrix spray method suitable for MALDI-MSI imaging

Abstract

The application discloses a 9-AA matrix spraying method suitable for MALDI-MSI imaging, and comprises the following steps: adding an ethanol aqueous solution and trifluoroacetic acid into 9-aminoacridine, and uniformly mixing to obtain a 9-AA matrix; after air bubbles in a spraying instrument are discharged, spraying parameters are set; the 9-AA matrix is injected into the spraying instrument; when a nozzle temperature reaches a set value and the matrix is normally sprayed from the nozzle, spraying of the matrix on a selected area is started; and MALDI-MSI imaging is performed on the sprayed sample. Compared with the prior art, the 9-AA matrix is uniformly attached to the surface of a tissue slice after the spraying condition of the matrix is optimized, the molecular imaging effect is smooth and coherent, and therefore, a good MALDI-MSI imaging effect is obtained.

Description

Technical Field

[0001] This invention belongs to the field of MALDI-MSI imaging, and specifically relates to a 9-AA matrix spraying method suitable for MALDI-MSI imaging. Background Technology

[0002] Matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI-MSI) is an advanced mass spectrometry imaging technique that combines substance identification and imaging capabilities. It can directly provide molecular maps containing information on the abundance and spatial distribution of biomolecules in tissues. Developed since the 1980s, this technique has expanded its application from macromolecules such as proteins, peptides, and nucleic acids to the detection of small molecule metabolites such as amino acids, lipids, and neurotransmitters. Simultaneously, novel matrices, including inorganic materials such as carbon, silicon, and metal nanomaterials, as well as novel organic molecules, have been developed, significantly improving the detection sensitivity, mass resolution, and imaging resolution of MALDI-MSI. While these new matrices enhance the sensitivity of substance detection, it is difficult to achieve uniform adhesion to the sample surface, thus affecting the imaging results of MALDI-MSI.

[0003] 9-AA (9-aminoacridine) is a commonly used matrix in MALDI-MSI imaging. However, due to its tendency to crystallize, 9-AA is difficult to distribute uniformly on conductive slides, affecting MALDI-MSI imaging results. Therefore, there is an urgent need for a spraying method suitable for improving the dispersion of the 9-AA matrix on conductive slides, in order to improve MALDI-MSI imaging and better provide in-situ information from tissue sections. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide a 9-AA matrix spraying method for MALDI-MSI imaging. This invention involves preparing a 9-AA matrix and setting spraying parameters such as air pressure, nozzle temperature, number of spraying cycles, matrix flow rate, and nozzle movement speed during the spraying process. This allows the 9-AA matrix to adhere uniformly to the surface of tissue sections, thereby achieving good MALDI-MSI imaging results.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A 9-AA matrix spraying method suitable for MALDI-MSI imaging includes the following steps:

[0007] An 80% aqueous ethanol solution and trifluoroacetic acid were added to 9-aminoacridine, and the mixture was sonicated for 30 minutes to obtain a homogeneous 9-AA matrix.

[0008] After removing air bubbles from the HTX™-Sprayer-HTX Imaging matrix sprayer using a 50% methanol aqueous solution, the spraying parameters were set as follows: gas pressure was set to 10 PSI, nozzle temperature was set to 80℃, number of spray cycles was set to 10, matrix flow rate was set to 0.065 mL / min, nozzle moving speed was set to 1200 mm / min, nozzle moving interval was set to 1 mm, gas flow rate was set to 2 L / min, and drying time was set to 10 s.

[0009] The metering loop was rinsed twice with acetonitrile, and then the sonicated 9-AA matrix was injected.

[0010] Once the nozzle temperature reaches the set value and the substrate is sprayed out of the nozzle normally, the selected area will be sprayed with substrate.

[0011] MALDI-MSI imaging was performed using the sprayed sample.

[0012] The present invention has the following beneficial effects:

[0013] Compared to existing technologies where uneven matrix spraying affects MALDI-MSI imaging results, leading to the presence of numerous microparticles in some areas of the molecular imaging effect, this invention optimizes the matrix spraying conditions to ensure that the 9-AA matrix adheres evenly to the surface of the tissue section, resulting in a smooth and consistent molecular imaging effect that presents the original effect of the material, thereby achieving excellent MALDI-MSI imaging results. Attached Figure Description

[0014] Figure 1 Figure A shows the spray effect when the flow rate is set to 0.125 mL / min, the number of cycles is 4, and the air pressure is 10 PSI. Figure B shows the effect when Figure A is magnified 4 times. Figure C shows the effect when Figure A is magnified 8 times.

[0015] Figure 2 Figure A shows the spray effect when the flow rate is set to 0.05 mL / min, the number of cycles is 4, and the air pressure is 10 PSI. Figure B shows the effect when Figure A is magnified 4 times. Figure C shows the effect when Figure A is magnified 8 times.

[0016] Figure 3 Figure A shows the spray effect when the flow rate is set to 0.065 mL / min, the number of cycles is 4, and the air pressure is 5 PSI. Figure B shows the effect when Figure A is magnified 4 times. Figure C shows the effect when Figure A is magnified 8 times.

[0017] Figure 4Figure A shows the spray effect when the flow rate is set to 0.065 mL / min, the number of cycles is 4, and the air pressure is 10 PSI. Figure B shows the effect when Figure A is magnified 4 times. Figure C shows the effect when Figure A is magnified 8 times.

[0018] Figure 5 Figure A shows the spray effect when the flow rate is set to 0.065 mL / min, the number of cycles is 10, and the air pressure is 10 PSI. Figure B shows the effect when Figure A is magnified 4 times. Figure C shows the effect when Figure A is magnified 8 times.

[0019] Figure 6 Image showing the effect of spraying a slice of mouse brain. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0021] In addition, unless otherwise specified, the preparation processes in the following embodiments are all conventional methods in the prior art, and therefore will not be described in detail.

[0022] A 9-AA matrix spraying method suitable for MALDI-MSI imaging includes the following steps:

[0023] Take 100 mg of 9-aminoacridine sample; add 10 mL of 80% ethanol aqueous solution and 10 μL of trifluoroacetic acid to the 9-aminoacridine sample, and then place it in an ultrasonic instrument and sonicate for 30 min to obtain the prepared 9-AA matrix;

[0024] Use a 50% methanol aqueous solution to purge air bubbles from the HTX™-Sprayer-HTX Imaging matrix sprayer. Set the spray parameters, including air pressure, nozzle temperature, number of spray wheels, matrix flow rate, nozzle movement speed, nozzle movement interval, gas flow rate, and drying time.

[0025] The metering loop was rinsed twice with acetonitrile, and then the sonicated 9-AA matrix was injected.

[0026] Once the nozzle temperature reaches the set value and the substrate is sprayed out of the nozzle normally, the selected area will be sprayed with substrate.

[0027] MALDI-MSI imaging was performed on the sprayed samples to compare the results.

[0028] Table 1 shows the key parameters for substrate spraying:

[0029] Table 1

[0030] parameter numerical values air pressure 5-10 PSI Nozzle temperature 70-90℃ Number of spray wheels 3-10 times Matrix flow rate 0.05-0.125 mL / min Nozzle movement speed 1200mm / min Nozzle movement interval 1-3mm gas flow rate 2L / min Drying time 5-10s

[0031] Figure 1 Figure A shows the spray effect with a flow rate of 0.125 mL / min, 4 passes, an air pressure of 10 PSI, a nozzle temperature of 75℃, a nozzle moving speed of 1200 mm / min, a nozzle moving interval of 3 mm, a gas flow rate of 2 L / min, and a drying time of 5 s. Figure B is a magnified version of Figure A by 4 times, and Figure C is a magnified version of Figure A by 8 times. From Figure 1 It can be seen that the matrix sprayed by this parameter is denser on the left and sparser on the right. The effect is more obvious when viewed under a microscope. The uneven thickness of the matrix spray seriously affects the ionization of substances inside the tissue by the laser, resulting in serious distortion of the mass spectrometry imaging signal.

[0032] Figure 2 Figure A shows the spray effect with a flow rate of 0.05 mL / min, 4 passes, an air pressure of 10 PSI, a nozzle temperature of 90℃, a nozzle moving speed of 1200 mm / min, a nozzle moving interval of 3 mm, a gas flow rate of 2 L / min, and a drying time of 5 s. Figure B is a magnified version of Figure A by 4 times, and Figure C is a magnified version of Figure A by 8 times. From Figure 2 It can be seen that the matrix spraying effect was improved after optimizing the matrix flow rate, and the matrix in the left and right parts was relatively uniform. However, after magnification, it was found that some areas were darker in color and thicker, which still affected the mass spectrometry imaging.

[0033] Figure 3 Figure A shows the spray effect with a flow rate of 0.065 mL / min, 4 passes, an air pressure of 5 PSI, a nozzle temperature of 80℃, a nozzle moving speed of 1200 mm / min, a nozzle moving interval of 2 mm, a gas flow rate of 2 L / min, and a drying time of 10 s. Figure B is a magnified version of Figure A by 4 times, and Figure C is a magnified version of Figure A by 8 times. From Figure 3 It can be seen that the spray effect is uniform throughout the glass slide, but upon magnification, it is found that the internal gaps are relatively large, which affects mass spectrometry imaging.

[0034] Figure 4 Figure A shows the spray effect with a flow rate of 0.065 mL / min, 4 passes, an air pressure of 10 PSI, a nozzle temperature of 80℃, a nozzle moving speed of 1200 mm / min, a nozzle moving interval of 1 mm, a gas flow rate of 2 L / min, and a drying time of 10 s. Figure B is a magnified version of Figure A by 4 times, and Figure C is a magnified version of Figure A by 8 times. From Figure 4 It can be seen that after adjusting the air pressure and nozzle movement interval, the matrix spray density has increased, but there are still gaps in the amplification effect.

[0035] Figure 5Figure A shows the spray effect with a flow rate of 0.065 mL / min, 10 passes, an air pressure of 10 PSI, a nozzle temperature of 80℃, a nozzle moving speed of 1200 mm / min, a nozzle moving interval of 1 mm, a gas flow rate of 2 L / min, and a drying time of 10 s. Figure B is a magnified version of Figure A by 4 times, and Figure C is a magnified version of Figure A by 8 times. From Figure 5 It can be seen that by adjusting the nozzle movement interval to 1mm, the matrix spray density is increased, the spray effect is consistent after magnification, and the mass spectrometry imaging effect is greatly improved.

[0036] The operating parameters were: air pressure 10 PSI, nozzle temperature 70℃, 4 spray cycles, matrix flow rate 0.125 mL / min, nozzle movement speed 1200 mm / min, nozzle movement interval 3 mm, gas flow rate 2 L / min, and drying time 5 s. The spraying effect on mouse brain slices was as follows: Figure 6 A1 and B1 in the image, molecular m / z 158.9257, MALDI-MSI imaging effect as follows: Figure 6 A2 and B2 in the image, molecular m / z 200.9013, MALDI-MSI imaging effect as follows: Figure 6 A3 and B3.

[0037] After parameter optimization, the following parameters were obtained: gas pressure 10 PSI, nozzle temperature 80℃, number of spray cycles 10, matrix flow rate 0.065 mL / min, nozzle moving speed 1200 mm / min, nozzle moving interval 1 mm, gas flow rate 2 L / min, and drying time 10 s. The spraying effect on mouse brain slices was as follows: Figure 6 In C1 and D1, the molecular m / z is 158.9257. The MALDI-MSI imaging effect is as follows: Figure 6 C2 and D2, molecular m / z 200.9013 MALDI-MSI imaging effect as follows Figure 6 Middle C3 and D3.

[0038] from Figure 6 It can be seen that the adjusted matrix spraying method results in visibly uniform matrix spraying, and the mass spectrometry imaging is equally smooth. The left side shows obvious agglomeration in the spray, with a significant signal intensity enhancement in the agglomerated areas. The right side shows the improved spraying and imaging results. Compared to the method on the left, the optimized method makes the matrix spraying more stable and uniform, resulting in more realistic imaging, greatly improving the resolution of substances within the tissue, and enhancing the mass spectrometry imaging signal intensity.

[0039] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A 9-AA matrix spraying method suitable for MALDI-MSI imaging, characterized in that: Includes the following steps: An aqueous ethanol solution and trifluoroacetic acid were added to 9-aminoacridine and mixed thoroughly to obtain a 9-AA matrix. After expelling air bubbles from the sprayer, set the spray parameters; the spray parameters include air pressure, nozzle temperature, number of spray wheels, matrix flow rate, nozzle moving speed, nozzle moving interval, gas flow rate, and drying time; Inject the 9-AA matrix into the sprayer. Once the nozzle temperature reaches the set value and the matrix is ​​sprayed out of the nozzle normally, start spraying the selected area with the matrix. MALDI-MSI imaging was performed on the sprayed sample. The air pressure is set to 10 PSI; The nozzle temperature is set to 80℃; The number of spray cycles is set to 10. The matrix flow rate was set to 0.065 mL / min; The nozzle moving speed is set to 1200 mm / min; The nozzle movement interval is set to 1mm; The gas flow rate is set to 2 L / min; The drying time is set to 10 seconds.

Images