Identification method for active sulfur

Abstract

To provide an identification method of reactive sulfur which improves the accuracy of identification of reactive sulfur molecules in a sample.SOLUTION: An identification method of reactive sulfur comprises the steps of: marking reactive sulfur in a sample with an alkylating agent; decomposing the reactive sulfur by adding a reducing agent to a portion of the extract of the first step; applying the sample subjected to a reduction treatment and the sample not subjected to the reduction treatment after marking to an analyzing device and comparing the analysis results of both samples with each other to make a component of a signal that has disappeared by the reduction treatment a candidate of the active sulfur in the sample; confirming that the signal is the signal derived from the alkylating agent; and, after the confirmation, extracting and purifying the component with the signal from the sample to analyze the structure of the component.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for identifying active sulfur. [Background technology]

[0002] Although the physiological importance of reactive sulfur species (RSSs), such as cysteine ​​polysulfides (CysSSHs) and glutathione polysulfides (GSSHs), has been recognized, the chemical properties of polysulfides are not fully understood and have not been elucidated due to their reactivity or complex redox-active properties.

[0003] Furthermore, because active sulfur molecules are highly unstable due to their high reactivity, they are easily decomposed during the extraction process from samples. As a method for detecting active sulfur molecules without this decomposition, a method has been developed in which active sulfur molecules are converted into stable derivatives using alkylating agents.

[0004] Regarding this detection method, the method reported in the previous research in the following two non-patent documents involves converting active sulfur molecules into stable derivatives using alkylating agents (monobromobimane, N-ethylmaleimide, beta-(4-hydroxyphenyl)ethyl iodoacetamide), followed by detection using a mass spectrometer. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Ida, T.; Sawa, T.; Ihara, H.; Tsuchiya, Y.; Watanabe, Y.; Kumagai, Y.; Suematsu, M.; Motohashi, H.; 111, 7606-7611, doi:10.1073 / pnas.1321232111. [Non-patent document 2] Ida, T.; Wei, FY; Nishida, M.; Kumagai, Y.; Alam, MM; Ihara, H.; Sawa, T.; Matsunaga, T.; Kasamatsu, S., et al. Cysteinyl-tRNA synthetase governs cysteine ​​polysulfidation and mitochondrial bioenergetics. Nat Commun 2017, 8, 1177, doi:10.1038 / s41467-017-01311-y. Summary of the Invention [Problem to be solved by the invention]

[0006] The above method detects and quantifies specific reactive sulfur molecules such as cysteine ​​persulfide and glutathione persulfide, but it can only detect specific reactive sulfur molecules whose molecular weights and structures are unknown. Therefore, there is a problem in that it is not possible to comprehensively search for unknown reactive sulfur molecules.

[0007] In view of this, the present invention aims to provide a method for identifying active sulfur that improves the accuracy of identifying active sulfur molecules when searching for active sulfur contained in various samples such as food, animal and plant tissues, cells, fungi, viruses, and clinical samples such as plasma, serum, cerebrospinal fluid, and bronchial lavage fluid. [Means for solving the problem]

[0008] 1. A method for identifying active sulfur in a sample, comprising: (a) a first step of labeling active sulfur in the sample with an alkylating agent; (b) a second step of adding a reducing agent to a portion of the extract from the first step to decompose active sulfur; (c) a third step of applying the reduced sample and the labeled sample that has not been reduced to an analytical device, comparing the analysis results of both samples, and determining the components of the signal that have disappeared due to the reduction treatment as candidates for active sulfur in the sample; (d) a fourth step of confirming that the signal is derived from the alkylating agent; (e) after the confirmation, a fifth step of extracting and purifying the signal components from the sample and analyzing the structure of the components; The method for identifying active sulfur is carried out. [Effects of the Invention]

[0009] According to the present invention, a method for identifying active sulfur can be provided that improves the accuracy of identifying active sulfur molecules in a sample. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is an explanatory diagram showing a method for identifying active sulfur according to an embodiment of the present invention. [Figure 2] FIG. 2 is an explanatory diagram showing decomposition of active sulfur structures by reducing agent treatment according to an embodiment of the present invention. [Figure 3] FIG. 1 is an explanatory diagram showing confirmation of an active sulfur component signal using a stable isotope-labeled reagent according to an embodiment of the present invention. [Figure 4] 1 shows the results of an example of searching for unknown active sulfur components according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] (Advantages of the present invention compared to conventional identification methods) Conventional methods for detecting active sulfur molecules use alkylating agents (especially monobromobimane and N-ethylmaleimide) to convert active sulfur molecules into stable derivatives and then detect them, in order to prevent the active sulfur components from being easily decomposed during the extraction process from the sample. However, for active sulfur molecules with unknown molecular weights and structures, alkylating agents have the effect of promoting the hydrolysis of active sulfur molecules, making it difficult to detect highly reactive unknown active sulfur molecules when using these alkylating agents.

[0012] In particular, commercially available alkylating agents have the effect of promoting the decomposition of active sulfur structures, making it difficult to detect and identify highly reactive (unstable) active sulfur molecules contained in samples using existing technologies, and it was not possible to comprehensively search for unknown active sulfur molecules.

[0013] Therefore, in the present invention, tyrosine derivatives are used as alkylating agents to select candidate active sulfur molecules. The following tyrosine derivatives, such as TME-IAM (N-iodoacetyl l-tyrosine methyl ester), have the effect of alkylating the (terminal) thiol group of active sulfur molecules to form stable derivatives and inhibit the decomposition of the active sulfur structure. By utilizing these effects, the accuracy of the selection of candidate active sulfur molecules is improved. The tyrosine derivative is identified by the following chemical formula:

[0014] [ka]

[0015] In the chemical formula of the tyrosine derivative above, one of the ac is "OH" and the rest are "H", and d is C n H 2n α, α is "H" or "a halogen atom", the halogen atom is preferably "I", n is an integer of 1 to 5, and e is (NH) x (CO) y C z H 2z β, where x is 1 or 0, y is 1 or 0, z is an integer of 1 to 5, and β is "H" or a "halogen atom", and the halogen atom is preferably "I". Furthermore, the above tyrosine derivatives include the following compounds.

[0016] [ka]

[0017] [ka]

[0018] [ka]

[0019] By selectively detecting product ions derived from TME-IAM during mass spectrometry, it is possible to comprehensively search for unknown reactive sulfur molecules at once. Furthermore, by using stable isotope-labeled TME-IAM, it is possible to more accurately select candidates for reactive sulfur molecules.

[0020] 1-1 (Synthesis method of TME-IAM) 1 mmol of L-tyrosine methyl ester was dissolved in N,N-dimethylformamide, and 2.2 mmol of the dehydration condensation agent N,N'-dicyclohexylcarbodiimide and 1 mmol of iodoacetic acid were added, followed by stirring on ice for 2 hours and then at room temperature for 1 hour.

[0021] Reagents [Synthesis of N-iodoacetyltyrosine methyl ester (TME-IAM)] Tyrosine Methyl Ester (Tokyo Chemical Industry Co., Ltd.) 195.2 mg N,N-dimethylformamide (Katayama Chemical) 2 mL N,N'-dicyclohexylcarbodiimide (nacalai tesque) 453.8 mg Iodoacetic acid (nacalai tesque) 186.0 mg

[0022] 1-2 (Product confirmation) To confirm the synthesized TME-IAM, the reaction solution was diluted with tyrosine methyl ester solution and then analyzed for absorbance at 220 nm, 250 nm, and 275 nm using high-performance liquid chromatography (HPLC).

[0023] Reagents 0.1% formic acid Formic Acid (abt.99%)(FUJIFILM)1mL Ultrapure water 999mL Methanol for LC / MS (Kanto Chemical)

[0024] equipment Pump: PU-2085 plus (JASCO) Autosampler: AS-2051 plus (JASCO) Detector: MD-2010 plus (JASCO) Degasser: DG-2085-54 (JASCO) Gradient mixer: MX-2080-32 (JASCO) Column oven: CO-2065 plus (JASCO)

[0025] HPLC conditions A buffer: 0.1% formic acid B buffer: methanol Gradient: [Table 1] Flow rate: 1 mL / min Inflow volume: 5μL Column: Mightysil RP-18 GP 75-3.0 (5 μm) (Kanto Chemical)

[0026] 2-1 (Crude purification of TME-IAM) After adding 4 mL of methanol and 181 g of Wakogel 100C to the reaction solution, 6 mL of 0.1% formic acid was added while stirring. This was filled into an empty reservoir and filtered. After mixing with 1 mL of methanol and 1 mL of 0.1% formic acid, the mixture was added to the empty reservoir and the eluate was collected and used as the crude solution.

[0027] Reagents 0.1% formic acid Methanol (Kanto Chemical) Wakogel (trademark) 100C18 (Wako)

[0028] 2-2 (Confirmation of crude purification) To confirm the purification of TME-IAM, the crude solution was diluted and then analyzed for absorbance at 220 nm, 250 nm, and 275 nm under the same conditions as in 1-2 to confirm the recovery of the reaction product.

[0029] 2-3 (Confirmation of products in crude purified solution) To confirm the presence of TME-IAM in the purified solution, the crude purified solution was diluted, and the presence of TME-IAM was confirmed by mass spectrometry using an HPLC-tandem mass spectrometer (LC-MS / MS).

[0030] Reagents 0.1% formic acid Methanol for LC / MS (Kanto Chemical)

[0031] equipment Mass spectrometer: Xevo TQD(Waters) HPLC: AllianceHPLC e2695(Waters)

[0032] HPLC conditions A buffer: 0.1% formic acid B buffer: Methanol (for LC / MS) Gradient: [Table 2] Flow rate: 0.3 mL / min Inflow volume: 5 μL Column: Mightysil RP-18 GP 50-2.0 (5 μm) (Kanto Chemical)

[0033] MS Scan conditions ES+ Q1 scan range (m / z): 50-600 Time(min):0-9 Cone voltage(V):10-35

[0034] 3-1 (Purification of TME-IAM) The crude solution was injected into a preparative HPLC column and collected in 2-minute fractions. The resulting fractions were analyzed for absorbance at 220 nm, 250 nm, and 275 nm under the same conditions as in 1-2, confirming the isolation of TME-IAM.

[0035] Reagents 0.1% formic acid Methanol (Kanto Chemical)

[0036] equipment Pump: PU-2089 plus (JASCO) Detector: 875-UV (JASCO)

[0037] HPLC fractionation conditions A buffer: 0.1% formic acid B buffer: methanol Gradient: [Table 3] Flow rate: 3 mL / min Detection wavelength: 275 nm Inflow volume: 1 mL Column: CAPCELL PAK C18 UG80 5 μm (SHISEIDO)

[0038] (Procedure of the identification method of the present invention): Figure 1 The procedure for carrying out the method for identifying unknown active sulfur components will be explained in steps 1 to 5. First, in step 1, active sulfur molecules are labeled with an alkylating agent. The first step will be specifically explained below.

[0039] The samples used include food such as onions and garlic, or animal tissues that have been chopped into 2-3mm pieces, suspensions of bacteria or algae, or liquids such as blood.

[0040] First, the sample is homogenized using a Polytron homogenizer in 80% methanol containing a new alkylating agent, TME-IAM (final concentration 1 mM), in an amount 10 times the weight of the sample. The homogenized sample is then protected from light and incubated at 37°C for 30 minutes to label the active sulfur component and thiol compound with the alkylating agent.

[0041] Thus, the use of TME-IAM for labeling has the effect of alkylating active sulfur molecules to form stable derivatives. Furthermore, TME-IAM suppresses hydrolysis of the active sulfur structure, thereby preventing decomposition of the active sulfur molecules during the extraction process from the sample.

[0042] The sample labeled with active sulfur components and thiol compounds was then centrifuged (18,000 g, 4°C, 15 minutes), and the supernatant was collected. One-hundredth of the volume of the supernatant was added with 10% formic acid to terminate the alkylation reaction.

[0043] Next, the reaction mixture was centrifuged to reduce its volume to approximately 1 / 10 of its original volume, and crude purification was performed using an octadecyl silica gel (ODS) column (Wakogel 100C18) equilibrated with 0.1% formic acid. Specifically, the concentrated sample was applied to the column in its entirety and washed with 0.1% formic acid. The TME-IAM-labeled derivatives of activated sulfur molecules and thiol compounds were then eluted using 80% methanol containing 0.1% formic acid. The eluate was centrifuged to reduce its volume to approximately 1 / 10 of its original volume, completing the crude purification.

[0044] As additives to inhibit the decomposition of active sulfur molecules, compounds having a hydroxyphenyl group or a hydroxyl group, such as tyrosine, glycerol, and polyvinyl alcohol, are used, and all of these are commercially available.

[0045] Furthermore, the alkylating agent is not limited to TME-IAM, but may be an analogous structure (an iodoacetamide derivative having a hydroxyphenyl structure or a tyrosine structure) such as methyltyramine iodoacetamide, as shown in the chemical formula above.

[0046] Next, in the second step, the active sulfur is decomposed by treatment with a reducing agent. The second step will be specifically described below.

[0047] A portion (approximately 100 μl) of the extract extracted during the labeling of active sulfur molecules in the first step is mixed with 20 mM phosphate buffer (pH 7.0) containing 2-mercaptoethanol (final concentration 1%).

[0048] The mixture is then incubated (reacted) for 1 hour at room temperature in the dark to decompose the active sulfur components, resulting in two samples: one that has been treated with the reducing agent and one that has not.

[0049] Next, in the third step, the active sulfur components are detected using a mass spectrometer. The third step will be specifically described below.

[0050] The samples treated with the reducing agent prepared in the second step and the samples not treated with the reducing agent were subjected to mass spectrometry using a liquid chromatography tandem mass spectrometry (HPLC-MS / MS) in multiple reaction monitoring mode, which scans precursor ions one mass at a time and detects the product ion (m / z 136) derived from TME-IAM.

[0051] As shown in Figure 2, the analytical results of the two samples obtained using this mass spectrometer are compared. Based on the comparison, the component whose signal disappears upon treatment with a reducing agent is identified as a candidate for the active sulfur component contained in the sample. This determination is based on the fact that the signal of the TME-IAM-labeled derivative (m / z 543) of glutathione, a thiol compound, does not disappear even after treatment with a reducing agent, whereas the signal of the TME-IAM-labeled derivative (m / z 575) of glutathione persulfide, an active sulfur molecule, completely disappears upon treatment with a reducing agent.

[0052] In the fourth step, stable isotope-labeled drugs are used to confirm whether the signal components identified in the third step are signals originating from the TME-IAM. The fourth step is described in detail below.

[0053] To make this determination, a stable isotope-labeled alkylating agent with a mass increased by two masses is used. The candidate active sulfur compounds synthesized using this alkylating agent are homogenized again in 80% methanol containing TME-IAM, as in the first step, and subjected to mass spectrometry. This allows us to confirm whether the precursor ion and product ion signals of the candidate active sulfur compounds, each increased by two masses, are detected in the mass spectrometer at the same retention time as the signal of the candidate active sulfur compounds detected in the third step.

[0054] For example, in the case of glutathione persulfide, as shown in Figure 3, a non-stable isotope-labeled product (m / z 575) and a stable isotope-labeled product (m / z 577) with a mass two times greater were used as precursors, and product ions (m / z 136 and 138) derived from the TME-IAM were detected. Both signals were detected at the same retention time, indicating that the signals were derived from the TME-IAM.

[0055] Finally, in the fifth step, the structure of the active sulfur component is analyzed and identified. The fifth step will be specifically described below.

[0056] In the fourth step, after confirming that the signals of the candidate active sulfur components are signals derived from TME-IAM, the active sulfur components are extracted and purified from the sample.

[0057] The homogenization in 80% methanol containing TME-IAM, as performed in the first step, is repeated on a large sample. The homogenized sample is centrifuged, and the supernatant is collected and concentrated. After concentration, the sample is roughly purified using an ODS solid-phase extraction pretreatment column.

[0058] The crude solution obtained here is fractionated by preparative HPLC. Each fraction is then subjected to a mass spectrometer to purify the active sulfur components. The purification of the active sulfur components is carried out using a combination of multiple HPLC columns with different separation modes, such as a normal-phase column, an ion-exchange column, and a mixed-mode column, in an ODS solid-phase extraction pretreatment column.

[0059] Thereafter, the active sulfur components were structurally analyzed and identified using a high-resolution mass spectrometer and a nuclear magnetic resonance (NMR) spectrometer. The above is the procedure for carrying out the method for identifying active sulfur of the present invention.

[0060] (Example of searching for active sulfur components) As an example of the present invention, a test to search for unknown active sulfur molecules in garlic is described below. First, to prepare a sample, 5.0 g of garlic cloves were added to 25 ml of methanol (containing 0.8 mM TME-IAM and 10 mM Tris-HCl, pH 7.4), and the mixture was homogenized using a Polytron homogenizer.

[0061] The homogenized reaction mixture was then incubated in the dark at 37° C. for 1 hour, after which 250 μl of 10% formic acid was added to the reaction mixture to terminate the reaction.

[0062] The reaction-terminated solution was centrifuged (4°C, 13,040 g, 20 minutes). The supernatant was collected and centrifuged to a volume of approximately 4-5 ml. 1 ml of 100% methanol and 9 ml of 0.1% formic acid were added to the centrifuged solution, and the mixture was centrifuged again (4°C, 13,040 g, 20 minutes).

[0063] The centrifuged solution was loaded onto a reversed-phase solid-phase column (Wakogel 100C18, column size: 3.5 ml) that had been previously equilibrated with 0.1% formic acid. Then, 17.5 ml of 0.1% formic acid was loaded onto the solid-phase column to wash the column. 5 ml of ultrapure water was loaded onto the solid-phase column, and the column was washed again. 17.5 ml of 100% methanol was loaded onto this solid-phase column, and the eluate was collected.

[0064] The eluate was concentrated to approximately 1 ml by centrifugation and then centrifuged (4°C, 13,040 g, 20 minutes). After centrifugation, the supernatant was collected and analyzed by liquid chromatography mass spectrometry (LC-MS / MS).

[0065] 100 μl of the above sample was reduced by adding 15 μl of 1M Tris-HCl (pH 7.4), 15 μl of 2-mercaptoethanol, and 20 μl of ultrapure water. The mixture was incubated at room temperature for 30 minutes in the dark and then subjected to LC-MS / MS analysis. The test results are shown in Figure 4.

[0066] HPLC conditions Flow rate: 0.3ml Inflow volume: 10μl Column: Mightysil RP-18 GP 50-2.0 (5 μm)

[0067] HPLC Gradient Program [Table 4]

[0068] MS conditions [Table 5]

[0069] (Discussion on test results) Preliminary studies were conducted using glutathione persulfide and glutathione TME-IAM adduct standard substances prepared in vitro, and the product ions generated from the TME-IAM adduct were determined. Furthermore, it was confirmed that when each sample was reduced with 2-mercaptoethanol before mass spectrometry, the signal disappeared only for the glutathione persulfide TME-IAM adduct standard substance. These results demonstrate the validity of the present invention.

[0070] Furthermore, analysis of mouse liver using the method of the present invention detected several candidate reactive sulfur molecules. Similarly, analysis using stable isotope-labeled TME-IAM detected a signal with a mass increase of 2. This strongly suggests that the signal detected in this study may be an adduct formed between TME-IAM and an unknown reactive sulfur molecule.

[0071] (Method of using the present invention) The novel sulfur compound identification method of the present invention can be applied to the analysis of food, animal and plant tissues, cells, fungi, viruses, and clinical samples (plasma, serum, cerebrospinal fluid, bronchial lavage fluid, etc.), thereby contributing to the search for novel physiologically active substances or active sulfur molecules that could serve as biomarkers for diseases and illnesses.

[0072] The method of the present invention as described above includes any modifications, substitutions and improvements made in accordance with the spirit and principles of the present invention.

Claims

1. 1. A method for identifying active sulfur in a sample, comprising: (a) a first step of labeling active sulfur in the sample with an alkylating agent; (b) a second step of adding a reducing agent to a portion of the extract from the first step to decompose active sulfur; (c) a third step of applying the reduced sample and the labeled sample that has not been reduced to an analytical device, comparing the analysis results of both samples, and determining the signal components that have disappeared due to the reduction treatment as candidates for active sulfur in the sample; (d) a fourth step of confirming that the signal is derived from the alkylating agent; (e) after the confirmation, a fifth step of extracting and purifying the signal component from the sample and analyzing the structure of the component; A method for identifying active sulfur, comprising:

2. The method for identifying active sulfur according to claim 1, wherein the alkylating agent is TME-IAM or a similar structure thereof.

3. The method for identifying active sulfur according to claim 1, wherein the analytical device is a liquid chromatography tandem mass spectrometer (HPLC-MS / MS).

4. The method for identifying active sulfur according to claim 1, wherein in the fifth step, the extraction and purification is carried out using an ODS solid-phase extraction pretreatment column.

5. The method for identifying active sulfur according to claim 1, wherein the purified solution obtained by the extraction and purification is fractionated by preparative HPLC and applied to the analytical device.

6. The method for identifying active sulfur according to claim 1, wherein the structural analysis of the component is carried out using a high-resolution mass spectrometer and a nuclear magnetic resonance (NMR) spectrometer.

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