Method of detecting matsutake mushroom using mass spectrometer

Abstract

To enable high-sensitivity detection of Matsutake mushroom that might cause allergies contained in food ingredients and products even if the amount is minute.SOLUTION: A method of detecting Matsutake mushroom is provided, comprising extracting protein from a sample, obtaining an enzyme digest of the extracted protein using a protease, and analyzing the enzyme digest to detect at least one peptide selected from a group consisting of SEQ ID NOs: 1 and 2 using a mass spectrometer so as to qualitatively or quantitatively determine the presence or absence of Matsutake mushroom protein in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for detecting matsutake mushrooms using a mass spectrometer, which enables highly sensitive detection of even minute amounts of matsutake mushrooms that may cause food allergies when they are contained in food ingredients or products. [Background technology]

[0002] Matsutake (Tricholoma matsutake) is a mushroom of the Tricholoma genus in the Tricholomataceae family, and in Japan it is designated as a "substance equivalent to a specific allergen" that is recommended to be labeled as a substance that can cause food allergies (Food Labeling Standards, Notice No. 139 of the Food Safety and Health Agency, March 30, 2015).

[0003] Foods that may cause allergies may be contaminated unintentionally in minute amounts during production, distribution, or processing, so it is important for providers of food ingredients or products to conduct quality control to determine whether or not such contaminants are present.

[0004] Methods for testing for the presence or absence of contamination in specific foods include methods that use antigen-antibody reactions such as ELISA, Western blotting, and immunochromatography to detect characteristic proteins, and methods that use PCR to detect characteristic DNA base sequences.

[0005] Recently, a method has been reported for detecting peptides derived from proteins characteristic of specific foods using mass spectrometry. This technology can quantify the protein in the target raw material, and has the advantages of reducing false positive reactions that tend to occur when using antigen-antibody reactions, and of being able to simultaneously detect multiple items.

[0006] As prior art relating to the detection of matsutake mushrooms, for example, the following prior art has been disclosed. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jang-Han Kim, and Yeong-Hwan Han, Development of Specific Primer for Tricholoma matsutake; Mycobiology, 37(4): 317-319, 2009 [Non-patent document 2] Luying Shan, Dazhou Wang, Yinjiao Li, Shi Zheng, Wentao Xu, and Ying Shang, Identification of the Pol Gene as a Species-Specific Diagnostic Marker for Qualitative and Quantitative PCR Detection of Tricholoma matsutake; Molecules, 24(3): 24030455, 2019

[0008] On the other hand, this document focuses on detecting genes, and other methods are possible. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, an object of the present invention is to provide an analytical method using a mass spectrometer that can specifically and sensitively detect matsutake mushrooms, which may cause allergies, in food ingredients and products. [Means for solving the problem]

[0010] In order to achieve the above object, the present inventors focused on the amino acid sequence of the matsutake protein to be detected and conducted extensive research into a method for specifically and highly sensitively detecting matsutake. As a result, they discovered amino acid sequences characteristic of matsutake, and discovered that detecting these amino acid sequences makes it possible to specifically and highly sensitively detect matsutake, thereby completing the present invention. That is, the present invention first relates to the following items.

[0011] Section 1. A method for detecting matsutake, which comprises the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein using a protease, analyzing the enzymatic digest, and detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 and 2 using a mass spectrometer to qualitatively or quantitatively determine whether or not matsutake protein is present in the sample.

[0012] Next, as a method for detecting at least one or more peptides selected from the group consisting of SEQ ID NOs: 1 and 2, a method is preferred in which the peptides are analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) and at least one or more precursor-product ion pair transitions having specific m / z values ​​associated with specific amino acid sequences are monitored. That is, the present invention relates to the following item 2.

[0013] Section 2. A step of extracting proteins from a sample, a step of obtaining an enzymatic digest of the extracted proteins using a protease, and a step of analyzing the enzymatic digest by liquid chromatography tandem mass spectrometry (LC-MS / MS) to obtain the following: i) SEQ ID NO: 1, m / z values ​​of about 768 / 432, 768 / 618, or 768 / 547 ii) SEQ ID NO: 2, m / z values ​​of about 571 / 670, 571 / 885, or 571 / 783 and determining whether or not matsutake protein is present in a sample by monitoring at least one precursor-product ion pair transition having a specific m / z value associated with a specific amino acid sequence selected from the group consisting of:

[0014] Next, it is preferable to monitor two or more of the precursor-product ion pair transitions. That is, the present invention relates to the following item 3. Section 3. The method for detecting matsutake according to claim 2, further comprising a step of qualitatively or quantitatively determining whether or not matsutake protein is present in a sample by monitoring at least two or more precursor-product ion pair transitions having specific m / z values ​​associated with the specific amino acid sequence. [Effects of the Invention]

[0015] According to the present invention, LC-MS / MS analysis enables the detection of peptides derived from Matsutake proteins, enabling the implementation of quality control tests to determine whether Matsutake is present in or used in test food ingredients or test foods. This can also contribute to the prevention of allergies and the investigation of causative substances when allergic symptoms occur. [Brief explanation of the drawings]

[0016] [Figure 1] Peptide peaks derived from Matsutake proteins in chromatograms obtained from standard samples with known Matsutake concentrations [Figure 2] A calibration curve was created by plotting the area of ​​peptides derived from Matsutake protein in a chromatogram obtained from a standard sample with a known Matsutake concentration against the known Matsutake protein concentration in the standard sample. [Figure 3] An exemplary chromatogram of a shiragayu sample containing no matsutake [Figure 4]An exemplary chromatogram containing peptides derived from Matsutake protein obtained from a rice porridge sample spiked with Matsutake protein. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a method for detecting minute amounts of Matsutake protein present in test samples such as food raw materials and processed foods. Specifically, the method comprises the steps of extracting protein from the test sample, obtaining an enzymatic digest of the extracted protein with a protease, and analyzing the enzymatic digest by LC-MS / MS to obtain a chromatogram of the target peptides. A preferred embodiment of the method according to this embodiment is described below.

[0018] Proteins can be extracted from test samples using a buffer solution containing a surfactant or a commercially available protein analysis kit.

[0019] Preferably, the protein extract from the test sample is further reduced and alkylated to block thiol groups.

[0020] The sample prepared as described above is treated with a protease. Examples of protease used in the method of the present invention include trypsin and chymotrypsin, with trypsin being preferred. Treatment conditions may be appropriately selected depending on the type of enzyme. The enzyme treatment degrades the target protein to generate multiple peptides.

[0021] The resulting enzymatic digests are preferably analyzed by LC-MS / MS after removing the surfactant and purifying using a reversed-phase solid-phase column.

[0022] The peptide sequences analyzed in LC-MS / MS are as follows: SEQ ID NO: 1 IGTDDQTSVADPTSK Sequence number 2 GVTTLPLGVER Although various methods can be used to detect these peptides derived from Matsutake, a mass spectrometer is used in the present invention. Among these, methods using liquid chromatography are particularly preferred, such as methods using LC-MS and LC-MS / MS. In particular, it is preferable to analyze the obtained enzymatic digest by LC-MS / MS after removing the surfactant and purifying it using a reversed-phase solid-phase column.

[0023] In addition, a standard sample with a known concentration of matsutake-derived protein can be treated in the same way as the test sample, analyzed by LC-MS / MS, and a calibration curve can be created to quantitatively analyze matsutake protein.

[0024] The type of test sample to be used in the matsutake detection method of the present invention is not particularly limited. Examples of test samples include food ingredients and processed foods. Food ingredients include food ingredients that are intentionally produced without matsutake, but are not included in a food ingredient production factory that handles matsutake. Processed foods include confectioneries, noodles, powdered soups, liquid soups, hot-air-dried or freeze-dried ingredients, and various prepared foods containing these processed foods. Other examples include processed foods that are intentionally produced without matsutake, but are not included in a food production factory that handles matsutake. Furthermore, when producing processed foods that do not contain matsutake after producing processed foods that contain matsutake, careful cleaning of the food production equipment is essential, with the removal of matsutake residue in mind. To verify the effectiveness of this cleaning method and the presence or absence of matsutake residue in the food production equipment, wiped samples from the production equipment can also be used as test samples.

[0025] Example The present invention will be described in more detail below using examples, but the present invention should not be construed as being limited to these examples. Furthermore, the present invention can be modified appropriately without departing from the gist of the present invention.

[0026] Example 1 Analysis of standard samples with known matsutake protein concentrations To verify the quantitative capability of the LC-MS / MS method for detecting Matsutake mushrooms of the present invention, standard samples with known Matsutake protein concentrations were analyzed and a calibration curve was created.

[0027] Protein was extracted from store-bought matsutake mushrooms using MPEX PTS Reagents (60 mM SDC SLS / 50 mM TEAB) (GL Sciences), and the total protein concentration was determined using a 2-D Quant Kit (Cytiva) to serve as a standard sample.

[0028] Of the prepared standard sample, 40 μg of protein was placed in a 2.0 mL low-adsorption polypropylene tube, and 1000 μg of egg-derived ovalbumin and 100 μg of bovine albumin were added to bring the total solution volume to 700 μL.

[0029] 70 μL of 1M TEAB and 28 μL of 1M DTT were added, and the mixture was left to stand at 75°C for 15 minutes, then at room temperature for 30 minutes. Next, 56 μL of 1M iodoacetamide solution prepared with distilled water was added, and the mixture was left to stand at room temperature in the dark for 45 minutes, after which 28 μL of 1M DTT was added (reduction / alkylation).

[0030] After adding 10 μL of a trypsin solution derived from bovine pancreas adjusted to 20 mg / mL with 0.1% formic acid, the mixture was left standing overnight at 37°C to perform enzymatic digestion of the Matsutake standard sample.

[0031] Formic acid was added to the resulting enzyme digest to make it acidic, and then ethyl acetate was added to remove the surfactant contained in the extract solution by liquid-liquid partitioning. This removal procedure was repeated three times.

[0032] The solution after removing the surfactant was concentrated using a centrifugal evaporator, and after adding 0.1% formic acid, the residue was purified using a C18 reversed-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase.

[0033] The refined solution was dried using a centrifugal evaporator, dissolved in 0.1% formic acid containing 5% acetonitrile, and a dilution series with a concentration range of 1.25 - 20 μg / mL in terms of the concentration in the sample of the total protein from shiitake mushrooms was prepared and analyzed by LC-MS / MS.

[0034] <LC-MS / MS instrument> LC section: ExionLC AD system (SCIEX) MS / MS section: QTRAP (registered trademark) 6500+ system (SCIEX) <LC conditions> Analysis column: YMC-Triart C18, particle size 3 μm, 100 x 2.1 mm id. (YMC) Column temperature: 40 °C Column flow rate: 0.3 mL / min Eluent A: 0.1% formic acid; Eluent B: acetonitrile containing 0.1% formic acid Gradient: 0 min (B: 5%) → 16 min (B: 40%) → 18 min (B: 95%) → 23 min (B: 95%) → 23.1 min (B: 5%) → initialization <Mass spectrometry conditions> Ionization: electrospray ionization method Polarity: positive Spray voltage: 5500 V

[0035] The sequences and MRM transitions of the peptide fragments derived from the shiitake mushroom protein to be detected are shown in Table 1.

[0036]

Table 1

[0037] The chromatogram when analyzing a standard sample with a concentration of 1.25 μg / mL as the total protein concentration from shiitake mushrooms is illustrated in Figure 1 (peptide sequence: IGTDDQTSVADPTSK (SEQ ID NO: 1), Q1: 767.9, Q3: 432.2).

[0038] Figure 2 shows an example of a calibration curve under the same detection conditions as Figure 1. A good calibration curve with R2: 0.998 was obtained in the range of 1.25 to 20 ppm, calculated as the concentration of matsutake total protein in the sample.

[0039] Example 2 Matsutake protein spike test in processed foods To examine the applicability of the LC-MS / MS method for detecting matsutake mushrooms of the present invention to processed foods, we analyzed white rice porridge containing no matsutake mushrooms to which a matsutake protein standard sample had been added at 10 ppm.

[0040] 1 g of a matsutake-free white rice porridge sample was weighed into a 50 mL polypropylene centrifuge tube, and the matsutake protein standard sample used in Example 1 was added to give a total matsutake protein concentration of 10 ppm.

[0041] 30 μL of ethylenediaminetetraacetic acid (EDTA) prepared at 100 mg / mL with 1N sodium hydroxide solution was added.

[0042] 9 mL of the extraction solution used in Example 1 was added, and the mixture was shaken at 90 to 110 rpm overnight to extract the protein.

[0043] The mixture was centrifuged at 10,000×g at 4°C for 30 minutes, and 700 μL of the supernatant was collected in a 2.0 mL low-adsorption polypropylene tube.

[0044] The subsequent procedures were carried out in the same manner as in Example 1, and the final undiluted solution was analyzed by LC-MS / MS.

[0045] Figure 3 shows a chromatogram of a porridge sample containing no matsutake mushrooms, and Figure 4 shows a chromatogram of a sample containing a matsutake protein standard sample added to a product content of 10 ppm (peptide sequence: IGTDDQTSVADPTSK (sequence number 1), Q1: 767.9, Q3: 432.2).

[0046] The peak to be detected was observed only when the matsutake protein standard sample was added.

Claims

1. A method for detecting matsutake, comprising the steps of extracting protein from a sample, treating the extracted protein with a protease to obtain an enzymatic digest, analyzing the enzymatic digest, and detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 and 2 using a mass spectrometer to qualitatively or quantitatively determine whether or not matsutake protein is present in the sample.

2. A step of extracting proteins from a sample, a step of treating the extracted proteins with a protease to obtain an enzymatic digest, and a step of analyzing the enzymatic digest by liquid chromatography tandem mass spectrometry (LC-MS / MS) to obtain the following: i) SEQ ID NO: 1, m / z values ​​of about 768 / 432, 768 / 618, or 768 / 547 ii) SEQ ID NO: 2, m / z values ​​of about 571 / 670, 571 / 885, or 571 / 783 and a step of qualitatively or quantitatively determining whether or not a matsutake protein is present in a sample by monitoring at least one precursor-product ion pair transition having a specific m / z value associated with a specific amino acid sequence selected from the group consisting of:

3. The method for detecting matsutake according to claim 2, further comprising a step of qualitatively or quantitatively determining whether or not matsutake protein is present in a sample by monitoring at least two or more precursor-product ion pair transitions having specific m / z values ​​associated with the specific amino acid sequence.

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