Method of detecting kiwi fruit using mass spectrometer

Abstract

To enable high-sensitivity detection of kiwi fruit that might cause allergies contained in food ingredients and products even if the amount is minute.SOLUTION: A method of detecting kiwi fruit 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 of SEQ ID NO: 1 using a mass spectrometer so as to qualitatively or quantitatively determine the presence or absence of kiwi fruit protein in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for detecting kiwifruit using a mass spectrometer, which enables highly sensitive detection of even trace amounts of kiwifruit, which may cause food allergies, when contained in food ingredients, products, etc. [Background technology]

[0002] Kiwifruit (Actinidia deliciosa, Actinidia chinensis, etc.) is a plant of the genus Actinidia in the family Actinidiaceae. In Japan, it is designated as a "substance equivalent to a specific raw material" 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 kiwifruit, for example, the following prior art has been disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 4385262 [Non-patent literature]

[0008] [Non-Patent Document 1] Seung-Man Suh, Saet-Byul Park, Mi-Ju Kim, and Hae-Yeong Kim, Simultaneous detection of fruit allergen-coding genes in tomato, apple, peach and kiwi through multiplex PCR; Food Sci. Biotechnol., 28(5): 1593-1598, 2019. [Non-patent document 2] Yi-Chen Shih 1, Jhih-Ting Hsiao 2, Fuu Sheu, Feasibility of Utilizing Stable-Isotope Dimethyl Labeling in Liquid Chromatography-Tandem Mass Spectrometry-Based Determination for Food Allergens-Case of Kiwifruit; Molecules., 24(10): 1920, 2019.

[0009] Of the above prior art, Non-Patent Document 2 is a method using mass spectrometry, but does not disclose the detection sensitivity for the total protein content derived from raw materials, which is considered important in the labeling system in Japan. Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, an object of the present invention is to provide an analytical method using a mass spectrometer that can specifically and highly sensitively detect kiwifruit, which may cause allergies, in food ingredients and products. [Means for solving the problem]

[0011] In order to achieve the above object, the present inventors have focused on the amino acid sequences of the kiwifruit allergen proteins to be detected and have conducted extensive research into a method for specifically and sensitively detecting kiwifruit. As a result, they have found amino acid sequences characteristic of kiwifruit, and have found that detecting these amino acid sequences enables specific and highly sensitive detection of kiwifruit, thereby completing the present invention. That is, the present invention first relates to the following items.

[0012] Section 1. A kiwifruit detection method comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein using a proteolytic enzyme, analyzing the enzymatic digest, and detecting the peptide consisting of sequence number 1 using a mass spectrometer to qualitatively or quantitatively determine whether or not kiwifruit protein is present in the sample.

[0013] Next, a preferred method for detecting the peptide consisting of SEQ ID NO: 1 is to analyze it by liquid chromatography tandem mass spectrometry (LC-MS / MS) and monitor at least one precursor-product ion pair transition having a specific m / z value associated with a specific amino acid sequence. That is, the present invention relates to the following item 2.

[0014] 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 430 / 159, 430 / 573, 430 / 474, 430 / 701, 430 / 260, 859 / 216, 859 / 287, 859 / 386, or 859 / 600 and determining whether or not a kiwifruit 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:

[0015] 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 kiwifruit detection method of claim 2, further comprising a step of qualitatively or quantitatively determining whether or not a kiwifruit 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]

[0016] The present invention enables the detection of kiwifruit protein-derived peptides by LC-MS / MS analysis, thereby enabling quality control testing to determine whether the kiwifruit is present in or used in test food ingredients or test foods, and can also contribute to the prevention of allergies and the investigation of causative substances when allergic symptoms occur. [Brief explanation of the drawings]

[0017] [Figure 1] Kiwifruit protein-derived peptide peaks in chromatograms obtained from standard samples with known concentrations of kiwifruit [Figure 2]A calibration curve was prepared by plotting the area of ​​peptides derived from kiwifruit protein in a chromatogram obtained from a standard sample with a known kiwifruit concentration against the known kiwifruit protein concentration in the standard sample. [Figure 3] An exemplary chromatogram of a white rice porridge sample without kiwifruit [Figure 4] An exemplary chromatogram containing peptides derived from kiwifruit protein obtained from a white rice porridge sample spiked with kiwifruit protein. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a method for detecting trace amounts of kiwifruit protein present in a test sample, such as a food ingredient or processed food. Specifically, the method includes 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.

[0019] Proteins can be extracted from test samples using a buffer solution containing a surfactant or a commercially available protein analysis kit.

[0020] Preferably, the protein extract from the test sample is further reduced and alkylated to block thiol groups.

[0021] 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.

[0022] The resulting enzymatic digests are preferably analyzed by LC-MS / MS after removing the surfactant and purifying using a reversed-phase solid-phase column.

[0023] The peptide sequences analyzed in LC-MS / MS are as follows: SEQ ID NO: 1 SAGAVVDIK Although various methods can be used to detect these kiwifruit-derived peptides, the present invention utilizes a mass spectrometer. 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.

[0024] In addition, a standard sample with a known concentration of kiwifruit-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 kiwifruit protein.

[0025] The type of test sample used in the kiwifruit 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 intentionally produced without kiwifruit in a food ingredient production factory that handles kiwifruit. 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 intentionally produced without kiwifruit in a food production factory that handles kiwifruit. Furthermore, when producing processed foods that do not contain kiwifruit after producing processed foods containing kiwifruit, thorough cleaning of the food production equipment is essential, with the removal of kiwifruit residue in mind. To verify the effectiveness of this cleaning method and to confirm the presence or absence of kiwifruit residue in the food production equipment, wiped samples from the production equipment can also be used as test samples.

[0026] 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.

[0027] Example 1 Analysis of kiwifruit standard samples with known protein concentrations To verify the quantitative capability of the kiwifruit detection method by LC-MS / MS of the present invention, standard samples with known concentrations of kiwifruit protein were analyzed and a calibration curve was prepared.

[0028] Proteins were extracted from kiwifruit purchased from a store 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.

[0029] 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.

[0030] 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).

[0031] 10 μL of bovine pancreas trypsin solution adjusted to 20 mg / mL with 0.1% formic acid was added, and the mixture was left standing overnight at 37°C to perform enzymatic digestion of the kiwifruit standard sample.

[0032] 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.

[0033] The solution after surfactant removal was concentrated using a centrifugal evaporator, and after adding 0.1% formic acid, purification was performed using a C18 reversed-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase.

[0034] The purified solution was dried using a centrifugal evaporator, dissolved in 0.1% formic acid containing 5% acetonitrile, and a dilution series with a concentration conversion of 1.25 - 20 μg / mL in the sample of kiwifruit total protein was prepared and analyzed by LC-MS / MS.

[0035] <LC-MS / MS device> LC section: ExionLC ADsystem (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

[0036] The sequences and MRM transitions of the kiwifruit protein-derived peptide fragments to be detected are shown in Table 1.

[0037]

Table 1

[0038] FIG. 1 shows an example of a chromatogram obtained by analyzing a standard sample with a kiwifruit total protein concentration of 1.25 μg / mL (peptide sequence: SAGAVVDIK (SEQ ID NO: 1), Q1: 430.2, Q3: 573.4).

[0039] Figure 2 shows an example of a calibration curve under the same detection conditions as Figure 1. A good calibration curve with R2: 0.990 was obtained in the range of 1.25 to 20 ppm, calculated as the total protein concentration in the kiwifruit sample.

[0040] Example 2 Kiwifruit protein spike test in processed foods To examine the applicability of the kiwifruit detection method of the present invention using LC-MS / MS to processed foods, a kiwifruit protein standard sample was added to 10 ppm of kiwifruit-free white rice porridge and analyzed.

[0041] 1 g of a kiwifruit-free white rice porridge sample was weighed into a 50 mL polypropylene centrifuge tube, and the kiwifruit protein standard sample used in Example 1 was added to give a total kiwifruit protein concentration of 10 ppm.

[0042] 30 μL of ethylenediaminetetraacetic acid (EDTA) prepared at 100 mg / mL with 1N sodium hydroxide solution was added.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Figure 3 shows a chromatogram of a white rice porridge sample that does not contain kiwi fruit, and Figure 4 shows a chromatogram of a sample to which a kiwi fruit protein standard sample was added at 10 ppm (peptide sequence: SAGAVVDIK (sequence number 1), Q1: 430.2, Q3: 573.4).

[0047] The target peak was observed only when the kiwifruit protein standard sample was added.

Claims

1. A kiwifruit detection method 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 NO: 1 using a mass spectrometer to qualitatively or quantitatively determine whether or not kiwifruit 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 430 / 159, 430 / 573, 430 / 474, 430 / 701, 430 / 260, 859 / 216, 859 / 287, 859 / 386, or 859 / 600 and qualitatively or quantitatively determining whether or not a kiwifruit 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. and a step of qualitatively or quantitatively determining whether or not a kiwifruit 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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