CN115753956A - Biological brain tissue DESI mass spectrum imaging processing method - Google Patents

Abstract

A DESI mass spectrum imaging processing method for biological brain tissue belongs to the technical field of mass spectrum detection. The imaging processing method comprises the following steps: the method comprises the steps of firstly taking animal brain tissue, freezing and slicing to obtain brain tissue slices, then adhering the brain tissue slices on a glass slide, freezing and storing, then taking the glass slide adhered with the brain tissue slices, carrying out DESI mass spectrometry imaging, processing scanning data through data processing software, deriving an ion imaging graph and a mass spectrogram, and finally comparing mass spectrometry information with mass spectrometry information of compounds in a biological database to screen out potential neurochemical substances. The method has high repeatability, does not need complex sample pretreatment, does not use a matrix, has high ionization efficiency, can be used for the distribution analysis of the micromolecular neurochemical substances in animal brain tissues, provides a quick and effective visual analysis method for the research of the spatial distribution and the spatial metabolism of the micromolecular neurochemical substances in the brain, and has good application value.

Description

Biological brain tissue DESI mass spectrum imaging processing method

Technical Field

The invention belongs to the technical field of mass spectrometric detection, and particularly relates to a DESI (Desi mass spectrometric imaging) processing method for biological brain tissue, which is used for analyzing micromolecular neurochemical substances and metabolite distribution thereof in the biological brain tissue.

Background

Mass Spectrometry Imaging (MSI) technology is a novel molecular imaging research approach that combines mass spectrometry with molecular imaging. Compared with the traditional imaging technology, the mass spectrometry imaging technology has more special specificity and higher accuracy, can directly and quickly analyze a sample, does not need extraction, derivation and marking, can provide molecular structures of various compounds, can reflect the space distribution condition of each molecule, has qualitative and quantitative analysis capability, and is gradually applied to the fields of pharmaceutical analysis, biomedicine, material characterization and the like (Yang, Y, C.et al.Univ.chem.2020,35,47-53 Yang, P, Y.et al.Sci.Sin.Vitae,2020,50, 1237-1255. Mass spectrometry imaging techniques can be classified into: secondary Ion Mass Spectrometry (SIMS), matrix Assisted Laser Desorption Ionization (MALDI), and desorption electrospray ionization (DESI), wherein SIMS and MALDI-MS require ionization under vacuum (Luo, Z, g.et al.sci.sin.chimi.2014,44,795-800, zhang, q, y.et al.anal.instrument.2018, 5, 1-10). And SIMS imaging techniques require appropriate modification of the sample surface to improve the analytical capabilities of molecular imaging.

The neurochemical is a chemical that exerts a neuroactivity in the nervous system, and may be an amino acid, a biogenic amine, a neuropeptide, or the like. Neurochemicals are involved in a variety of biophysical processes and play an important role in the regulation of central nervous, immune, endocrine, and other systems. The brain is the most complex organ of mammals, and is divided into more than two hundred fine subregions, and different regional structures exert respective biological functions. The content change of the neurochemical substances in the brain tissue and the distribution of different brain regions are closely related to various diseases and can be used as key indexes for clinical diagnosis, screening and treatment of the diseases (Brichta L, et al. Trends neurosci.2013,36,543-554, zhao L. Et al. Chromanogr.B.2015, 988,59-65, kenche V B. Et al. Inorg. Chem.2013,52, 4303-4318.

Commonly used methods for detecting neurochemicals include derivatization treatments such as ultraviolet, fluorescence, electrochemistry, etc. to improve the response signals of the neurochemicals, but derivatization operations are cumbersome, accuracy and repeatability are difficult to control, and larger errors are brought to detection (Ganesana m.et al. Ham.chem.2017, 89,314-341 bucher e.et al. Chem.2015,8,239-261, weii b.et al. Pharm.biomed.anal.2014,88, 416-422. At present, the application of mass spectrometry imaging technology in the field of brain neurochemistry research mainly focuses on the analysis of biological macromolecules such as proteins, peptides, lipids and the like by MALDI-MS. And the matrix effect exists in MALDI-MS I imaging analysis, which makes the spectrum analysis more complicated and limits the application of MALDI-MS I imaging analysis in the aspect of small molecule compound imaging analysis. DESI is a normal pressure open type soft ionization mass spectrometry technology, does not need complex sample pretreatment, does not use matrix, has high ionization efficiency, and can realize the research on the spatial distribution and the associated change of endogenous and exogenous small molecular substances in biological tissue samples (Feng, B, S.et al.Univ.chem.2013,28,1-8, wang, X, Q.et al.chem.J.Chin.Univ.2020,41,2673-2680, lostun, D.et al.chem.2015,87, 3286-3293.

Disclosure of Invention

The invention aims to provide a DESI mass spectrometry imaging method for detecting spatial distribution of small molecule neurochemicals and metabolites thereof in rat whole brain. The imaging method has high repeatability, does not need to carry out pretreatment such as homogenization, derivatization and the like on brain tissues, is used for directly freezing and slicing detection, and is simple to operate. Provides a quick and effective visual analysis method for the research of the spatial distribution and the spatial metabolism of the small-molecule neurochemical substances in the brain.

The second purpose of the invention is to provide a DESI mass spectrometry imaging data processing method, which is characterized in that mass spectrometry information of potential neurochemical substances in an imaging graph and a mass spectrogram of different ions is derived and is compared with mass spectrometry information of compounds in a biological database, so as to screen out the potential neurochemical substances in a brain tissue slice.

The technical scheme adopted by the invention for realizing the aim is as follows.

The invention provides a biological brain tissue DESI mass spectrum imaging processing method, which comprises the following steps:

taking animal brain tissue, freezing and slicing to obtain brain tissue slices;

adhering the brain tissue slices on a glass slide, and freezing and storing;

step three, performing DESI mass spectrum imaging on the frozen and stored glass slide adhered with the brain tissue slice in a positive ion mode and a negative ion mode;

step four, processing the DESI mass spectrum imaging scanning data by data processing software, and then deriving an ion imaging graph and a mass spectrogram;

and step five, comparing the mass spectrum information of the mass spectrogram with the mass spectrum information of the compound in the biological database, and screening out the neurochemical substance.

Further, in the first step, the freezing condition is-15-25 ℃.

Further, in the first step, before freezing, the animal brain tissue is rinsed and then is dried by suction.

Further, in the first step, the animal is a rat; further, the rat is a Wistar rat.

Further, in the first step, the slice thickness is 8-11 μm.

Further, in the first step, the slicing equipment is a slicer, and the internal temperature of the slicer is-18 to-25 ℃.

Furthermore, in the second step, the freezing condition is-78 to-83 ℃.

Further, in the third step, the spraying solvent adopted by the DESI mass spectrum imaging is 0.08-0.12 wt% of formic acid methanol solution.

Further, in the third step, before the imaging of the DESI mass spectrum, the mass spectrum is corrected by sodium formate.

Further, in the third step, 0.15-0.22 μ g/mL Leucine Enkephalin (LE) is added into the spray solvent adopted by the DESI mass spectrometry imaging, and the mass spectrometry is corrected in real time in the DESI mass spectrometry imaging process.

Further, in the third step, the parameters of the DESI mass spectrometry imaging are as follows: the DESI ion source adopts a positive ion mode and a negative ion mode, the collection range is m/z 50-1000, the collection is carried out in a sensitivity mode, the capillary voltage is 3.7-4.2 kV, the ion source temperature is 130-170 ℃, the nitrogen flow rate is 0.42-0.48 mL/min, and the spray solvent flow rate is 1-4 mu L/min.

Further, in the fourth step, the data processing software is hdiging v1.5.

Further, in the fifth step, the biological database is HMDB (http:// www.hmdb.ca).

Compared with the prior art, the invention has the following beneficial effects:

(1) The DESI mass spectrum imaging processing method of brain tissue of the invention is a new application direction of the mass spectrum imaging technology in the brain neurochemical research, which not only focuses on the analysis of biomacromolecules such as protein, peptide and lipid by MALDI-MS technology in the field, widens the application field of the mass spectrum imaging technology, and provides a rapid and effective visual analysis method for the research of the space distribution and space metabolism of small molecule neurochemical substances in the brain.

(2) The DESI mass spectrum imaging processing method of the brain tissue does not need complex pretreatment processes such as homogenization, derivatization and the like of the brain tissue, is directly used for frozen slice detection, is simple to operate, reduces the complexity of an experiment, reduces errors, greatly improves the accuracy of the experiment, and is quick and effective.

(3) The DESI mass spectrum imaging processing method of brain tissue of the invention provides a new choice of spray solvent, so that the mass spectrum signal intensity of each neurochemical substance is improved to different degrees.

(4) Compared with other methods, the DESI mass spectrometry imaging processing method of the brain tissue does not need to modify the surface of a sample to improve the analysis capability of molecular imaging, does not have matrix interference, has higher ionization efficiency, accurate detection result, higher sensitivity and good repeatability, and can realize the detection and analysis of the spatial distribution of small-molecule neurochemical substances in the brain.

(5) According to the DESI mass spectrum imaging processing method of the brain tissue, disclosed by the invention, the data is subjected to fine imaging analysis through HDI mapping software, and different ion imaging graphs and mass spectrograms are derived. And the mass spectrum information of the potential neurochemical substances is innovatively compared with a biological database HMDB, so that the method for screening the potential neurochemical substances is provided, and the data processing process can be used as a novel mass spectrum imaging data processing process to be applied to the analysis of brain tissue small molecule neurochemical substances.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph of the effect of formic acid addition to the spray solvent on the signal intensity of the mass spectrum of a neurochemical in example 1, (A) methanol, (B) 0.08wt% to 0.12wt% methanoic acid;

FIG. 2 is a graph of the effect of formic acid addition to the spray solvent on the imaging of neurochemicals in example 1;

FIG. 3 is a DESI image of Gln in three adjacent brain sections in positive ion mode with 0.08wt% to 0.12wt% methanoic acid methanol spray as in example 1.

Detailed Description

For a further understanding of the invention, the following description of the preferred embodiments of the invention is provided, but it is to be understood that the description is only intended to illustrate further features and advantages of the invention, and not to limit the scope of the claims.

As introduced by the background art, the application of mass spectrometry imaging technology in the field of brain neurochemistry research is mainly focused on the analysis of biological macromolecules such as proteins, peptides and lipids by MALDI-MS, and the matrix effect exists in MALDI-MSI imaging analysis, so that the map analysis becomes more complex, and the application of the MALDI-MSI imaging technology in the aspect of imaging analysis of small molecular compounds is limited.

In view of the above, the invention utilizes a DESI mass spectrometry imaging method, widens the application of mass spectrometry imaging in the field of biological small molecules, and establishes a method for analyzing the distribution of small molecule neurochemicals in brain tissues based on a DESI technology. The method comprises the following steps:

taking animal brain tissue, freezing and slicing to obtain brain tissue slices;

adhering the brain tissue slices on a glass slide, and freezing and storing;

step three, performing DESI mass spectrum imaging on the frozen and stored glass slide adhered with the brain tissue slice in a positive ion mode and a negative ion mode;

step four, processing the DESI mass spectrum imaging scanning data by data processing software, and then deriving an ion imaging graph and a mass spectrogram;

and step five, comparing the mass spectrum information of the mass spectrogram with the mass spectrum information of the compound in the biological database, and screening out potential neurochemical substances.

In the technical scheme, in the first step, the freezing condition is preferably-15 to-25 ℃. However, the present invention is not limited to this, and any freezing temperature that can ensure that the brain tissue of the animal is not damaged may be used.

In the above technical scheme, in the step one, the animal is not particularly limited, and what animal needs to be analyzed and used. In this embodiment, the animal is a rat, and preferably a Wistar rat. It should be noted that, in order to ensure the accuracy of the analysis, it is considered to use repeated analysis with increasing number of animals, such as 10 Wistar rats, each half of male and female, which are detected separately.

In the above technical solution, in the first step, the slice thickness is preferably 8 to 11 μm. However, it should be noted that the thickness of the slice can be determined by those skilled in the art according to actual needs, and is not particularly limited.

According to the technical scheme, in the first step, the slicing device is a slicer, and the internal temperature of the slicer is preferably-18 to-25 ℃. However, it should be noted that the person skilled in the art may obtain slices by other known methods, and the temperature may be other freezing temperatures that ensure that the brain tissue of the animal is not damaged.

According to the technical scheme, in the first step, before freezing, the animal brain tissue is rinsed and then is dried by suction, the rinsing is performed by using ice-cold physiological saline at the temperature of minus 17 to minus 23 ℃, and the filter paper is dried by suction.

In the technical scheme, in the second step, the freezing condition is preferably-78 to-83 ℃. However, the present invention is not limited to this, and any freezing temperature that can ensure that the brain tissue of the animal is not damaged may be used.

In the third step, the spraying solvent adopted by the DESI mass spectrum imaging is 0.08-0.12 wt% of formic acid methanol solution. The selection of the spray solvent is not conventional in the art, and allows mass spectrum signal intensity of each neurochemical substance to be improved to different degrees.

In the above technical scheme, in the third step, the parameters of the DESI mass spectrometry imaging are as follows: the DESI ion source adopts a positive ion mode and a negative ion mode, the collection range is m/z 50-1000, the collection is carried out in a sensitivity mode, the capillary voltage is 3.7-4.2 kV, the ion source temperature is 130-170 ℃, the nitrogen flow rate is 0.42-0.48 mL/min, and the spray solvent flow rate is 1-4 mu L/min.

In the third step, sodium formate is preferably used for correcting the mass spectrum before the imaging of the DESI mass spectrum; leucine Enkephalin (LE) of 0.15-0.22 mu g/mL can be added into the spraying solvent, and the mass spectrum is corrected in real time in the process of DESI mass spectrum imaging analysis.

In the above technical solution, in step four, the data processing software is preferably hdiging v1.5. It should be noted that other data processing software suitable for use with the present invention may be employed by those skilled in the art.

In the fifth step of the technical scheme, the biological database is HMDB (http:// www.hmdb.ca). It is to be noted that other suitable biological databases may be used by those skilled in the art.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, apparatuses, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.

The present invention is further illustrated by the following examples.

Example 1

Firstly, killing a rat with broken head, immediately taking a whole brain on an ice bench, rinsing the whole brain in ice-cold physiological saline, sucking the physiological saline by filter paper, and freezing at-15 to-25 ℃ overnight;

sticking the whole brain on a slice mop by using glue, manufacturing a tissue slice on a CM3050S cryomicrotome (LEICA company) after the glue is completely dried, flatly placing the slice on a glass slide at the temperature of-18 to-25 ℃ and the slice thickness of 8 to 11 mu m, and storing the slice in a refrigerator at the temperature of-78 to-83 ℃ to be detected;

thirdly, performing mass spectrometry imaging acquisition analysis on the frozen brain tissue slices by using a Waters synapse G2-Si (Waters corporation) mass spectrometry imaging system, correcting the mass spectrometry by using sodium formate before the acquisition analysis is started, adding 0.15-0.22 mu G/mL Leucine Enkephalin (LE) into a spray solvent, and correcting the mass spectrometry in real time in the experimental process;

adjusting an included angle between an ion source spray nozzle of a mass spectrometry imaging system and a sample glass slide to be 53-62 degrees, wherein the vertical distance between the ion spray nozzle and the sample glass slide is 1.3-1.7 mm, the horizontal distance between the ion spray nozzle and a mass spectrometry collection unlet pipe orifice is 3-6 mm, the vertical distance between the mass spectrometry collection unlet pipe orifice and the sample glass slide is 0.3-0.8 mm, and the scanning speed is 480-510 mu m/sec;

the specific mass spectrometry parameters set were: the DESI ion source adopts a positive ion mode and a negative ion mode, wherein the collection range is m/z 50-1000, the collection is carried out in a sensitivity mode, the capillary voltage is 3.7-4.2 kV, the ion source temperature is 130-170 ℃, the nitrogen flow rate is 0.42-0.48 mL/min, and the spray solvent flow rate is 1-4 mu L/min;

step four, performing fine imaging analysis on mass spectrum imaging scanning data of rat brain tissue slices in a positive and negative ion mode by applying HDIMAging v1.5, deriving imaging graphs and mass spectrograms of 2002 different ions, comparing mass spectrum information of potential neurochemical substances in the imaging graphs and the mass spectrum information of compounds in an HMDB database, and screening 25 neurochemical substances, wherein the table 1 shows that:

TABLE 1 potential neurochemicals in rat brain tissue sections in Positive and negative ion modes

To examine the effect of different spray solvents on the signal intensity of neurochemicals in brain tissue sections. Taking GABA, ser and Asp as examples, selecting methanol, 47-63 wt% methanol water and pure water as a fog solvent for scanning and acquisition, comparing the relative intensities of ions, the signal intensities of GABA, ser and Asp in positive and negative ion modes are better than that of 47-63 wt% methanol water under the condition of methanol spray solvent, and the signal intensity in pure water is very weak. In 25 screened neurochemicals, signals of 16 compounds were detected in total under methanol spray conditions. Molecular ion signals of GABA, ser and Asp are detected in the positive ion mode and the negative ion mode, and the detected signal strength is higher in the positive ion mode. Gly, DA, NE, DHPG, 5-HT, tyr, HVA, E, DOMA, MHPG, meldonin were detected only in the positive ion mode, while Ach, DOPAC, VMA were detected only in the negative ion mode. No molecular ion signal is detected in positive and negative ion modes of Histamine, tau, gln, glu, phe, 3-MT, 5-HIAA and Try under the condition of methanol spray solvent.

And investigating the influence of 0.08-0.12 wt% formic acid added into the spray solvent on the detection signal intensity of the mass spectrum of the neurochemical substance. When 0.1wt% of methanoic acid was used as the spray solvent, 25 neurochemicals detected their molecular ion signals in both positive and negative ion modes, and the peak intensities of each molecular ion were higher in the positive ion mode, with the signal intensity changes as shown in fig. 1. After formic acid was added to the spray solvent, 9 more neurochemicals, histamine, tau, gin, glu, phe, 3-MT, 5-HIAA, and Try were detected than when formic acid was not added. The originally detected signals of 16 neurochemicals are also greatly improved, the signal intensity is improved by 30 to 45 times (DHPG) at the highest in the positive ion mode, and the signal intensity is improved by 15 to 25 times (GABA) at the highest in the negative ion mode. Therefore, the mass spectrum signal intensity of the formic acid on each neurochemical substance is improved to different degrees, and the signal intensity is improved more obviously in the positive ion mode.

According to the determined spray solvent, the influence of the spray solvent on the imaging effect of the neurochemical mass spectrum is further considered, in a positive ion mode, methanol with or without formic acid is used as the spray solvent, two continuous adjacent sections of rat brain tissue are scanned and data are acquired, an imaging graph of the neurochemical is obtained after the treatment of HDI mapping software, and by taking GABA, ser and Asp as examples, as shown in FIG. 2, the imaging intensity of the neurochemical can be improved to different degrees by adding formic acid.

The repeatability of mass spectrometry imaging technology is the key to the reliability of the application of the technology. In the positive ion mode, 0.08-0.12 wt% of methanoic acid is used as a spray solvent, and imaging data acquisition and processing are carried out on three continuous adjacent sections of rat brain tissue. Taking Gln as an example, the spatial distribution of Gln detected in three adjacent brain tissue slices is shown in FIG. 3, and Matlab software is used to calculate the similarity of imaging pictures to be 85.6% -93.1%, which indicates that the mass spectrometry imaging method has high repeatability.

As demonstrated by FIGS. 2 and 3, DESI mass spectrometry can effectively represent the distribution of neurochemicals in the brain, where GABA and Asp are mainly distributed in the cerebral cortex, ser is distributed in the whole brain, and Gln is mainly concentrated around the hippocampus.

It should be understood that the above embodiments are only examples for clarity of description, and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither necessary nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims (10)

1. A DESI mass spectrum imaging processing method of biological brain tissue is characterized by comprising the following steps:

taking animal brain tissue, freezing and slicing to obtain brain tissue slices;

adhering the brain tissue slices on a glass slide, and freezing and storing;

step three, performing DESI mass spectrum imaging on the frozen and stored glass slide adhered with the brain tissue slice in a positive ion mode and a negative ion mode;

step four, processing the DESI mass spectrum imaging scanning data by data processing software, and then deriving an ion imaging graph and a mass spectrogram;

and step five, comparing the mass spectrum information of the mass spectrogram with the mass spectrum information of the compound in the biological database, and screening out the neurochemical substance.

2. The DESI mass spectrometry processing method of claim 1, wherein in the first step, the freezing condition is-15 to-25 ℃, the animal is rat, the slicing apparatus is a microtome, the internal temperature of the microtome is-18 to-25 ℃, and the slice thickness is 8 to 11 μm.

3. The method as claimed in claim 1, wherein in step one, the animal brain tissue is rinsed and then dried by blotting before freezing.

4. The method for DESI mass spectrometry imaging of biological brain tissue according to claim 1, wherein the freezing condition in step two is-78 to-83 ℃.

5. The method for DESI mass spectrometry imaging of biological brain tissue according to claim 1, wherein in the third step, the spraying solvent used for DESI mass spectrometry imaging is 0.08-0.12 wt% formic acid methanol solution.

6. The method for DESI mass spectrometry of biological brain tissue according to claim 1, wherein in step three, the mass spectrometry is corrected with sodium formate before DESI mass spectrometry.

7. The method for DESI mass spectrometry imaging of biological brain tissue according to claim 1, wherein in the third step, 0.15-0.22 μ g/mL leucine enkephalin is added into the spray solvent used for DESI mass spectrometry imaging, and mass spectrometry is corrected in real time during DESI mass spectrometry imaging.

8. The method for processing DESI mass spectrometry of biological brain tissue according to claim 1, wherein in the third step, the parameters of DESI mass spectrometry imaging are: the DESI ion source adopts a positive ion mode and a negative ion mode, wherein the collection range m/z is 50-1000, the collection is carried out in a sensitivity mode, the capillary voltage is 3.7-4.2 kV, the ion source temperature is 130-170 ℃, the nitrogen flow rate is 0.42-0.48 mL/min, and the spray solvent flow rate is 1-4 mu L/min.

9. The DESI mass spectrometry processing method for biological brain tissue according to claim 1, wherein in step four, the data processing software is HDI magic v1.5.

10. The method for DESI mass spectrometry of biological brain tissue according to claim 1, wherein in step five, the biological database is HMDB.

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