JP7290164B2 - Method for measuring reduced glutathione distribution on living tissue and method for acquiring diagnostic data - Google Patents

Description

The present invention relates to a method for measuring the distribution of reduced glutathione (GSH) on living tissue using mass spectrometry imaging (MSI).

GSH is an oxidative stress marker, and is an important substance whose quantitative evaluation is being attempted to evaluate various diseases. As one of the evaluation methods, quantitative visualization of GSH on tissue sections, especially technology development using MSI, has been eagerly awaited. .

Aesif et al., Protocols for the Detection of S-Glutathionylated and S-Nitrosylated Proteins In Situ Methods in Enzymology, Volume 474 Baez et al., Mass spectrometry in studies of protein thiol chemistry and signaling: Opportunities and caveats FreeRadicalBiologyandMedicine80(2015)191-211 Nazari et al., Quantitative mass spectrometry imaging of glutathione in healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) Analyst, 2018, 143, 654

An object of the present invention is to provide a method for accurately measuring the GSH distribution on a sample using MSI.

Under such circumstances, the present inventors have made intensive studies, and as a result, when detecting GSH distributed in a sample by multi-step MSI, a step of spraying an SH group oxidation protective agent on the sample,
The present inventors have found that the above problems can be solved by subjecting the matrix agent-coated sample to multistage MSI after carrying out the step of applying the matrix agent to the sample sprayed with the SH group oxidation protective agent. In addition, the present invention is based on the above-mentioned novel findings of the present inventors. Based on this finding, the present inventors conducted various studies on an SH group oxidation protective agent for derivatizing GSH, a matrix agent for assisting ionization, and the like, and completed the present invention. Accordingly, the present invention provides the following terms:
Section 1. (1) a step of spraying an SH-group oxidation protective agent onto a sample;
(2) applying a matrix agent to a sample sprayed with an SH-group oxidation protective agent; A method for measuring reduced glutathione distribution.

Section 2. 3. The method of paragraph 1, wherein the multi-step mass spectrometry imaging is in positive mode.

Item 3. Item 3. The method of Item 2, wherein the matrix agent comprises α-cyano-4-hydroxycinnamic acid.

Section 4. 3. The method of paragraph 1, wherein the multi-step mass spectrometry imaging is in negative mode.

Item 5. Item 5. The method according to Item 4, wherein the matrix agent contains at least one selected from the group consisting of 2,5-dihydroxybenzoic acid and 1,5-diaminonaphthalene.

Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the SH group oxidation protective agent contains at least one selected from the group consisting of N-substituted maleimide derivatives, pyridine derivatives and 2-nitrobenzoic acid derivatives.

Item 7. A kit for measuring reduced glutathione distribution on living tissue by multi-step mass spectrometry imaging, comprising an SH group antioxidant and a matrix agent.

Item 8. 8. The kit of paragraph 7 for using positive mode multi-step mass spectrometry imaging, wherein the matrix agent comprises α-cyano-4-hydroxycinnamic acid.

Item 9. Item 8. The kit according to item 7 for using negative mode multi-step mass spectrometry imaging, wherein the matrix agent contains at least one selected from the group consisting of 2,5-dihydroxybenzoic acid and 1,5-diaminonaphthalene.

Item 10. Item 9. The kit according to any one of Items 7 to 9, wherein the SH group oxidation protective agent contains at least one selected from the group consisting of N-substituted maleimide derivatives, pyridine derivatives, and 2-nitrobenzoic acid derivatives.

In contrast, Non-Patent Document 1 describes a method for detecting S-glutathionylated cysteine residues on tissue sections, in which unmodified cysteine residues are blocked using N-ethylmaleimide (NEM). It merely describes immersing a tissue section in a NEM solution, and neither a method for detecting GSH nor a method for measuring a target substance using mass spectrometry (MS) is described. In addition, Non-Patent Document 2 only describes subjecting a protein or the like whose thiol group is modified with NEM to MS, and does not describe a method for measuring the distribution of a target substance using MSI. In addition, Non-Patent Document 2 does not describe the application of GSH to MS. Then, Non-Patent Document 3 only describes evaluation of GSH by negative mode MSI, and does not describe a method for measuring the distribution of GSH derivatized on tissues using MSI.

According to the present invention, it is possible to provide a method for accurately measuring the GSH distribution on a sample using MSI. In addition, conventionally, most of the cases tried to detect GSH itself instead of multi-stage MSI. Under such circumstances, the present inventors applied a matrix agent to the sample after derivatizing the SH group of GSH on the tissue in a method including the above steps, and then subjected the matrix agent-applied sample to multistep MSI. It was found that the GSH distribution can be measured with high accuracy while suppressing the oxidation of GSH during ionization. Therefore, such an effect of the present invention cannot be expected from the prior art.

1 shows a schematic of an exemplary embodiment of the invention; Reaction schemes of GSH and NEM, and m/z before and after fragmentation for GSH and NEM-derivatized GSH are shown. The results of Example 1 are shown. The results of Example 2 are shown.

1. In one embodiment of the method for measuring GSH distribution , the present invention comprises the steps of: (1) spraying a sample with an SH-group oxidation protective agent;
(2) Applying a matrix agent to a sample sprayed with an SH group antioxidant, and (3) Subjecting the sample to which the matrix agent is applied to multi-step MSI. provide a way to A schematic of an exemplary embodiment of the invention is shown in FIG.

1.1. Step of Spraying SH Group Antioxidant In the method of the present invention, first, the sample is sprayed with an SH group antioxidant ((1) in FIG. 1).

The sample to be measured is not particularly limited as long as it contains GSH, and examples thereof include biological tissue sections of animals or plants, processed foods, rocks, industrial products, and the like. When a biological tissue section is used as a sample, the animal from which the biological tissue section is collected is not limited. Examples include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep, hamsters, guinea pigs, mammals such as goats; non-mammalian animals such as nematodes, fruit flies and zebrafish; and plants such as Arabidopsis thaliana, rapeseed, rice and wheat. Processed foods include, for example, bread, fish paste products, and processed meat products. Industrial products include, for example, electronic substrates. In the present invention, the sample is preferably a biological tissue section, more preferably a mouse, rat or human biological tissue section, and still more preferably a human biological tissue section. When a biological tissue section is used as a sample, the tissue from which the biological tissue section is collected is not particularly limited. Uterus, brain, kidney, prostate, ovary, spleen, lymph node, thyroid, heart, skeletal muscle, intestine, esophagus, adrenal gland, testis, adipose tissue, eyeball, spinal cord, inner ear, thymus, blood vessel, hair, bone, fetus, whole body etc.

The method of preparing the sample to be subjected to the method of the present invention is not particularly limited. It can be prepared by drying frozen sections. Although the thickness of the sample is not particularly limited, it can be set, for example, in the range of 1 to 500 μm, preferably 4 to 100 μm. The shape of the sample is preferably a flat plate, but is not particularly limited. When the sample is flat, the area of the flat portion is not particularly limited, but can be set within the range of 5 μm×5 μm to 26 mm×76 mm, for example.

The SH group oxidation protective agent is not particularly limited as long as it can bind to SH of GSH and protect it. Examples include N-substituted maleimide derivatives, pyridine derivatives, and 2-nitrobenzoic acid derivatives. be done. N-substituted maleimide derivatives include, for example, N-ethylmaleimide (NEM), N-methylmaleimide (NMM), N-propylmaleimide (NPM), N-benzylmaleimide (NBM) and the like, preferably NEM and the like. . Pyridine derivatives include 2-vinylpyridine (2-VP), dithiodipyridine (PDS) and the like. 2-nitrobenzoic acid derivatives include 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and the like. Among these SH group oxidation protective agents, N-substituted maleimide derivatives, particularly NEM, are preferred. These SH group oxidation protective agents can be used singly or in combination of two or more. In preferred embodiments of the invention, these SH-group oxidation protectants can be sprayed, for example, as a solution in a solvent. In such an embodiment, the solvent is not particularly limited as long as it does not interfere with the reaction for protecting GSH by the SH group oxidation protective agent, and may be appropriately selected according to the SH group oxidation protective agent to be used. water, ethanol, methanol, 2-propanol, acetonitrile and the like. These SH group oxidation protective agents can be used singly or in combination of two or more. In such an embodiment, the molar concentration of the SH group oxidation protective agent in the solution is not particularly limited, but can be set, for example, in the range of 0.1-100 mM, preferably 10-500 mM.

The spraying method is also not particularly limited, but for example, a sample may be placed on a slide glass, and a solution appropriately containing an SH-group oxidation protective agent may be sprayed onto the sample using an airbrush or the like. The amount of the SH group oxidation protective agent to ^be sprayed is not particularly limited as long as it is ^an amount that can protect SH of GSH when GSH is present in the sample. It can be set to spray 1 micromol to 100 micromoles, preferably 1 micromol to 10 micromoles, of the SH group oxidation protective agent per sample (1.0 cm×1.0 cm×8.0 μm). Although the entire amount of the SH-group oxidation protective agent may be sprayed at once, it is recommended to repeat the spraying and natural drying in multiple steps in order for the SH-group oxidation protective agent to efficiently adhere to and permeate the sample. preferable. Although the spraying time is not particularly limited, it can be set, for example, in the range of 1 to 120 minutes, preferably 15 to 60 minutes. The temperature at which the spraying step is performed is also not particularly limited, but can be set, for example, in the range of 0 to 60°C, preferably 10 to 30°C. The pressure of the air pump connected to the air spray during the spraying step is also not particularly limited, but can be set in the range of, for example, 0.01 to 0.2 MPa, preferably 0.1 to 0.15 MPa. The spraying step can form SH group-protected derivatives of GSH, thereby protecting GSH from oxidation. In Non-Patent Document 1, a tissue section fixed on a slide glass is derivatized with a NEM solution. In this method, a relatively simple method of immersing the entire tissue section in a NEM solution is employed in order to allow NEM to react with the cysteine residues of proteins that are insoluble in a solvent. On the other hand, the present invention includes a step of directly derivatizing GSH, which is a low-molecular-weight compound present on the sample surface. Since GSH present on the sample surface is easily dissolved in a solvent such as water, it is expected that GSH will be washed away by the NEM solution even if the method of Non-Patent Document 1 is used to measure GSH in the sample. In contrast, according to the present invention, GSH present on the sample surface can be directly derivatized by spraying an SH-group oxidation-protecting agent onto the tissue surface without washing away the GSH.

After the spraying step, the sample is subjected to the matrix agent coating step. Between the spraying step and the matrix agent application step, an incubation step is optionally performed in which the sample sprayed with the SH group oxidation protective agent is left to stand to ensure derivatization (protection reaction) of GSH in the sample. can be When incubation is performed, the temperature is not particularly limited, but can be set, for example, in the range of 4-60°C, preferably 10-30°C. Although the time is not particularly limited, it can be set, for example, in the range of 5 to 360 minutes, preferably 5 to 60 minutes. The pressure during incubation is also not particularly limited, but can be set, for example, in the range of 900-1200Pa, preferably 900-1100Pa. This step is preferably carried out in that the derivatization of GSH proceeds during the incubation step.

1.2. Step of Applying a Matrix Agent In the method of the present invention, the next step is to apply a matrix agent to the sample sprayed with the SH group oxidation protective agent to assist the ionization of the derivatized GSH in multistep mass spectrometry imaging. ((2) in FIG. 1).

The matrix agent is not particularly limited as long as it can be used for MALDI-MSI, but preferably, for example, one that can absorb laser light and assist the ionization of derivatized GSH. For example, when using a 355 nm laser beam, α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), 1,5-diaminonaphthalene (1,5-DAN) and the like. These matrix agents can be used singly or in combination of two or more.

In addition, the present inventors found that when performing multistep MSI in the positive mode, the MSI measurement of NEM-derivatized GSH can be performed more efficiently by using, for example, CHCA among matrix agents. The suitability of these particular matrix agents for measurements of GSH distribution in positive mode was hitherto unknown and unexpected from the prior art.

Furthermore, the present inventors performed MSI measurement of NEM-derivatized GSH in negative mode by using matrix agents such as DHB, 1,5-DAN, etc., even when performing multi-step MSI in negative mode. I have found that it can be done more efficiently. The suitability of these particular matrix agents for measuring GSH distributions in negative mode was hitherto completely unknown and unexpected from the prior art.

Although the thickness of the matrix agent to be applied to the sample is not particularly limited, it can be set, for example, in the range of 0.1 to 5.0 μm, preferably 1.0 to 2.0 μm.

The method of applying the matrix agent is not particularly limited, but depending on the properties of the matrix agent, for example, vapor deposition, spraying, a combination of vapor deposition and spraying, recrystallization after vapor deposition or spraying, etc., can be mentioned, and vapor deposition is preferred. is mentioned. The matrix agent application step is primarily a necessary step to assist the ionization of the derivatized GSH in subsequent multi-step mass spectrometry imaging. The coating step can be carried out by appropriately using an apparatus for coating a matrix agent, which is known in the technical field to which the present invention belongs.

1.3. Step of Multistep Mass Spectrometry Imaging (Multistep MSI) In the method of the present invention, the next step is to subject the sample coated with the matrix agent to multistep MSI.

Multi-step MSI is the product or precursor mass spectrum for ions selected by their mass-to-charge ratio (m/z) values, or mass spectra of precursor ions with specific m/z value increases or decreases, repeated two or more times. It is an MSI that uses the acquisition technique. In a typical embodiment, ions of a specific mass number among the ionized molecules collide with an inert gas (Ar, He, Xe, etc.) to generate product ions, which are secondary ions, The target substance is measured by detecting the product ion.

In an exemplary embodiment of the invention, the technique of MALDI (Matrix-Assisted Laser Desorption Ionization) is used. Specifically, a sample coated with a matrix agent is irradiated with a laser to excite the matrix and desorb and ionize the GSH derivative, which is the target substance. Ionized GSH derivatives are detected with a mass spectrometer as described above. This makes it possible to obtain the ionic strength and coordinates derived from the GSH derivative. By changing the irradiation position and angle of the laser and making measurements, it is possible to measure the relative amount of GSH at each location on the sample. Therefore, the distribution of GSH in the sample can be visualized.

In one embodiment of the invention, multi-stage MSI can be performed in positive mode. Since GSH has a thiol group and a carboxyl group, measurement in negative mode has conventionally been attempted when attempting to detect GSH by MSI. Therefore, it has not been known until now that the distribution of GSH can be suitably measured by performing multi-step MSI in the positive mode, and this is a matter that cannot be predicted from the prior art. Positive mode (positive ion mode) refers to a method of generating and detecting positive ions of a compound to be measured or a fragment thereof, and negative mode (negative ion mode) refers to a method of detecting negative ions of a compound to be measured or a fragment thereof. It refers to the method of generation and detection. This embodiment is characterized by ionizing derivatized GSH from a sample coated with a specific matrix agent by multi-stage MSI and measuring the ions in positive or negative mode.

The multi-step MSI process can be performed by appropriately using an apparatus for performing multi-step MSI known in the technical field to which the present invention belongs. Conditions such as detector voltage, irradiation diameter, and (laser) intensity vary depending on the type of apparatus and sample. For example, the detector voltage is preferably 0.5 to 2.1 kV, more preferably 1.00 to 2.10 kV. The irradiation diameter is preferably 5-100 μm, more preferably 5-25 μm. The (laser) intensity is 0 to 100 (Note: 0 in this case means that the laser high intensity memory in the multi-stage MSI device (typically iMScope (manufactured by Shimadzu Corporation) used in the present example) is 0. In other words, it means the weakest laser light intensity in the device.Similarly, the value of laser intensity means the value in the memory of the multi-step MSI device.), preferably 40.0 to 60.0. A multi-step MSI process can provide image data showing the distribution of GSH in the sample.
As described above, according to the method of the present invention, image data showing the distribution of GSH in a sample can be obtained. In addition, GSH is an antioxidant that exists in high concentrations in cells, and is known to contribute to protection against oxidative stress caused by active oxygen, free radicals, and the like. Related to this, it is also known that there are diseases that can be evaluated by changes in GSH. Specific examples include cancer; neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease; liver diseases such as hepatitis C and non-alcoholic steatohepatitis; and drug addiction. Therefore, image data showing the distribution of GSH in a sample obtained by the method of the present invention is useful for diagnosing the above diseases. Therefore, the method of the present invention can also be used as a diagnostic data acquisition method. Diseases that can be evaluated by changes in GSH include, but are not limited to, cancer, neurodegenerative diseases, immune diseases, drug addiction, and the like. Examples of cancer include lung cancer, colon cancer (rectal cancer, colon cancer), small intestine cancer, stomach cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, laryngeal cancer, skin cancer, uterine cancer, brain tumor, kidney cancer, and prostate cancer. solid tumors such as cancer, ovarian cancer, spleen cancer, thyroid cancer, sarcoma, esophageal cancer, breast cancer, bladder cancer; malignant lymphoma;

Moreover, as one embodiment of the present invention,
(1) a step of spraying an SH-group oxidation protective agent onto a sample;
(2) applying a matrix agent to a sample sprayed with an SH-group oxidation protective agent; A method of measuring a distribution comprising:
A method is provided in which the matrix agent contains at least one selected from the group consisting of DHB and 1,5-DAN.

In this embodiment, even when negative-mode multi-step MSI is used, a method for accurately measuring the GSH distribution on a sample using MSI is provided by using the above matrix agent. can provide.

Conditions other than performing multi-step MSI in negative mode and using the above as a matrix agent, such as measurement sample, type and amount of SH group oxidation protective agent, method of applying matrix agent, embodiment in multi-step MSI Conditions such as detector voltage, irradiation diameter, (laser) intensity, etc. in , can be the same as those described above in the embodiment in which multi-stage MSI is performed in positive mode.

In addition, the intensity information of the signal on the tissue surface in the MALDI-MSI measurement according to the present invention is reconstructed as a two-dimensional image based on the positional information of the optical microscope image taken in advance and converted into an MSI image. An image with a clear distribution of GSH is obtained.

(2. Kit)
In another embodiment, the present invention provides a kit for measuring GSH distribution on living tissue by positive mode multi-step MSI, comprising an SH group antioxidant and a matrix agent, and an SH group antioxidant and matrix agent. A kit for measuring GSH distribution on biological tissue by negative mode multi-step MSI, comprising:
A kit is provided in which the matrix agent contains at least one selected from the group consisting of CHCA, DHB and 1,5-DAN.

The meanings of the terms SH group oxidation protective agent, matrix agent, multistage MSI, etc. are the same as above. The kit of the present invention can be used for the above-described method for measuring GSH distribution and the like. In addition, the kit of the present invention can contain other components as needed. Other components include, for example, tools for collecting samples, syringes for transferring the SH group antioxidant and/or matrix agent to the device used in each step, positive control samples, negative control samples, and the like. , but not limited to. Written instructions or the like for carrying out the aforementioned method of the present invention may also be included.

An embodiment of the present invention will be specifically described below using examples, but the present invention is not limited to the following examples.

Abbreviations A description of the abbreviations used in this example follows:
MALDI-MSI: matrix assisted laser desorption/ionization-mass spectrometry imaging;
NEM: N-methylmaleimide;
CHCA: α-cyano-4-hydroxycinnamic acid; α-cyano-4-hydroxycinnamic acid GSH: glutathione;
GSH-NEM: NEM derivatized GSH
MS/MS: mass spectrometry/mass spectrometry; tandem mass spectrometry
Example 1
material and method
[Reagents for MALDI-MSI]
Methanol was purchased from Kanto Kagaku Co., Ltd., and NEM was purchased from Tokyo Chemical Industry Co., Ltd. CHCA was purchased from Merck (formerly Sigma-Aldrich).

[Animal tissue]
Tissue sections used in this example were prepared from C57BL/6J mice (female, 7 months old; purchased from Japan SLC, Inc.). All mouse handling and experiments were performed in accordance with the guidelines of the Tohoku University Graduate School of Medicine and the Animal Care and Use Committee.

[Tissue derivatization and sample preparation for MALDI-MSI]
Using a cryostat (CM3050S; Leica Microsystems), cryosections of 8 μm thickness were made from unfixed frozen mouse brain tissue, which were pasted onto indium tin oxide-coated glass slides (SI0100N; Matsunami Glass) and transferred to silica gel. dried in a 50 mL plastic tube containing Next, the tissue section on the slide glass was sprayed with a 100 mM 15% (v/v) methanol solution of NEM using an airbrush (Mr. hobby Procon boy FWA Platinum 0.2 double action; manufactured by GSI Cryos). Spraying and natural drying were repeated. The amount of spray per slide glass was 100 µL, and the needle stopper was adjusted so that it took about 30 minutes to spray the entire amount. After all the NEM solution was sprayed onto the glass slide, it was left at room temperature for 1 hour to complete the derivatization reaction of GSH contained in the tissue section. CHCA as a matrix was vapor-deposited on this sample to a thickness of 0.7 μm using an automatic pretreatment device iMLayer (manufactured by Shimadzu Corporation) to obtain a sample for MALDI-MSI. As a control experiment, a sample was prepared by vapor-depositing CHCA to a thickness of 0.7 μm on a section obtained in the same manner as described above, except that NEM derivatization was not performed.

[MALDI-MSI measurement method without MS/MS]
MALDI-MSI measurement was immediately performed on the sample after CHCA deposition using a mass microscope iMScope (manufactured by Shimadzu Corporation). MALDI-MSI measurement conditions without MS/MS are as follows. Ion species: positive ion, measurement range: m/z 300-315 (non-derivatized sample), m/z 400-500 (derivatized sample), integration times: 1 time/pixel, sample voltage: 3.50 kV, detector Voltage: 2.10 kV, MS stages: 1. The laser irradiation conditions are as follows. Irradiation count: 100 shots, Repetition frequency: 1000 Hz, Irradiation diameter: 2 (25 μm), Intensity: 48.0.

[MALDI-MSI measurement method using MS/MS]
The MALDI-MSI measurement conditions using MS/MS are as follows. Ion species: positive ion, measurement range: m/z 85-315 (non-derivatized sample), m/z 250-435 (derivatized sample), integration times: 1 time/pixel, sample voltage: 3.50 kV, detector Voltage: 2.10 kV, MS stages: 2, CID: ON, Precursor: m/z 308.09 (underivatized sample), 433.14 (underivatized sample), Width: 2.0000 amu, Energy: 50% (underivatized sample), 120 (derivatized sample), gas volume: 50%, isolation time: 20 msec, CID time: 30 msec. The laser irradiation conditions are as follows. Irradiation count: 100 shots, Repetition frequency: 1000 Hz, Irradiation diameter: 2 (25 μm), Intensity: 50.0.

[analysis]
The intensity information of the signal on the tissue surface in the MALDI-MSI measurement was reconstructed as a two-dimensional image based on the position information of the optical microscope image taken in advance, and the MSI image was obtained. Imaging MS Solution (manufactured by Shimadzu Corporation) was used as analysis software.

[result]
In the MALDI-MSI measurement without MS/MS, the GSH hydrogenated ion signal was observed at m/z 308.10 and the GSH-NEM hydrogenated ion signal was observed at m/z 433.14. In the MALDI-MSI measurement using MS/MS, the signal for the product ion with GSH as the precursor was observed at m/z 179.05, and the signal for the product ion with GSH-NEM as the precursor was observed at m/z 304.10 (Fig. 2, Figure 3 A-D). In the MALDI-MSI measurement without MS/MS of the underivatized sample, a strong signal derived from CHCA was observed throughout the measurement range, and no clear image of the tissue distribution of GSH was obtained (Fig. 3A). In the MALDI-MSI measurement using MS/MS of the non-derivatized sample and the MALDI-MSI measurement of the derivatized sample without using MS/MS, a tissue-specific signal was observed, but the intensity was low and a good image was not obtained. It was not obtained (Fig. 3 B, C). On the other hand, the MALDI-MSI measurement using MS/MS of the derivatized sample obtained a clear image of the distribution of GSH on the tissue with sufficient signal intensity. (Fig. 3D).

Example 2
Samples were prepared and pretreated in the same manner as in Example 1, except that CHCA was vapor-deposited to a thickness of 1.5 μm, and MALDI-MSI measurements using MS/MS of derivatized samples were performed. The results are shown in FIG. As shown in Fig. 4, when the thickness of CHCA was 1.5 µm and the laser intensity was 54, the signal intensity was further improved than when the thickness was 0.7 µm (Fig. 3D), indicating the distribution of GSH on the sample surface. became clearer.

The method of the present invention can provide a method for measuring the GSH distribution on living tissue with high accuracy. As described above, the present invention has high industrial applicability, particularly in technical fields such as medical treatment, because various diseases can be evaluated based on GSH fluctuations.

Claims (11)

(1) a step of spraying an SH-group oxidation protective agent onto a sample;
(2) applying a matrix agent to the sample sprayed with the SH group oxidation protective agent; and (3) subjecting the sample coated with the matrix agent to multi-step mass spectrometry imaging ,
In step (1), the SH group of reduced glutathione is derivatized on a biological tissue,
A method for measuring reduced glutathione distribution on living tissue by mass spectrometry imaging. 2. The method of claim 1, wherein multi-step mass spectrometric imaging is in positive mode. 3. The method of claim 2, wherein the matrix agent comprises alpha-cyano-4-hydroxycinnamic acid. 2. The method of claim 1, wherein the multi-step mass spectrometry imaging is in negative mode. 5. The method of claim 4, wherein the matrix agent comprises at least one selected from the group consisting of 2,5-dihydroxybenzoic acid and 1,5-diaminonaphthalene. The method according to any one of claims 1 to 5, wherein the SH group oxidation protective agent comprises at least one selected from the group consisting of N-substituted maleimide derivatives, pyridine derivatives and 2-nitrobenzoic acid derivatives. 7. The method according to any one of claims 1 to 6, wherein in the step of (1) spraying the SH group oxidation protective agent onto the sample, the spraying and the natural drying are repeated in multiple steps. an SH group oxidation protective agent for spraying onto a sample to derivatize the SH group of reduced glutathione on the biological tissue ;
a matrix agent for application to a sample in which the SH group of reduced glutathione is derivatized on a biological tissue;
A kit for measuring reduced glutathione distribution on living tissue by multi-step mass spectrometry imaging. 9. The kit of claim 8 for using positive mode multistep mass spectrometry imaging, wherein the matrix agent comprises α-cyano-4-hydroxycinnamic acid. 9. The kit according to claim 8, wherein the matrix agent contains at least one selected from the group consisting of 2,5-dihydroxybenzoic acid and 1,5-diaminonaphthalene, for use in negative mode multistep mass spectrometry imaging. The kit according to any one of claims 8 to 10, wherein the SH group oxidation protective agent contains at least one selected from the group consisting of N-substituted maleimide derivatives, pyridine derivatives and 2-nitrobenzoic acid derivatives.

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