Method of detecting apple using mass spectrometer

Abstract

To enable high-sensitivity detection of apple that might cause allergies contained in food ingredients and products even if the amount is minute.SOLUTION: A method of detecting apple 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 through 3 using a mass spectrometer so as to qualitatively or quantitatively determine the presence or absence of apple protein in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] Apples (Malus domestica) are a plant of the genus Malus in the family Rosaceae. In Japan, they are designated as "equivalent to specific ingredients" that are recommended to be labeled as substances that can cause food allergies (Food Labeling Standards, Notice No. 139 of the Food Safety and Health Administration, 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 apples, for example, the following prior art has been disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 4937305 [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] Satoshi Watanabe, Hiromu Taguchi, Yusuke Temmei, Takashi Hirao, Hiroshi Akiyama, Shinobu Sakai, Reiko Adachi, Atsuo Urisu, and Reiko Teshima, Specific Detection of Potentially Allergenic Peach and Apple in Foods Using Polymerase Chain Reaction; J. Agric. Food Chem., 60(9): 2108-2115, 2012.

[0009] On the other hand, the patent and non-patent documents are concerned with detecting genes, and other methods may also be possible. 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 sensitively detect apples, 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 focused on the amino acid sequences of apple proteins to be detected and conducted extensive research into a method for specifically and sensitively detecting apples. As a result, they discovered amino acid sequences characteristic of apples and found that detecting these amino acid sequences enables specific and highly sensitive detection of apples, leading to the completion of the present invention. That is, the present invention first relates to the following items:

[0012] Section 1. A method for detecting apples, comprising the steps of extracting proteins from a sample, obtaining enzymatic digests of the extracted proteins using protease, analyzing the enzymatic digests, and detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 3 using a mass spectrometer to qualitatively or quantitatively determine whether or not apple proteins are present in the sample.

[0013] Next, as a method for detecting at least one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 3, a method is preferred in which the peptides are analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) to monitor at least one or more precursor-product ion pair transitions having specific m / z values ​​associated with specific amino acid sequences. 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 516 / 820, 516 / 748, 516 / 213, or 516 / 661 ii) SEQ ID NO: 2, m / z value of about 476 / 606 iii) SEQ ID NO: 3, m / z values ​​of about 528 / 897, 528 / 737, 528 / 449, 528 / 624, 528 / 553, 528 / 234, 518 / 319, or 518 / 406 and determining whether or not an apple 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. and a step of qualitatively or quantitatively determining whether or not apple proteins are present in the 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 apple protein-derived peptides by LC-MS / MS analysis, thereby enabling quality control inspections to be carried out to determine whether the above-mentioned apples are present in or used in test food ingredients or test foods. This method 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] Peaks of peptides derived from apple proteins in chromatograms obtained from standard samples with known apple concentrations [Figure 2] A calibration curve was created by plotting the area of ​​peptides derived from apple protein in a chromatogram obtained from a standard sample with a known apple concentration against the known apple protein concentration in the standard sample. [Figure 3] An exemplary chromatogram of a rice porridge sample without apples [Figure 4] An exemplary chromatogram containing peptides derived from apple protein obtained from a rice porridge sample spiked with apple protein. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a method for detecting trace amounts of apple protein present in test samples such as raw food 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 this method 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: Sequence number 1 LVASGSGSIIK SEQ ID NO:2 AADGSVISCK Sequence ID No. 3 SAC[CAM]LAFGDSK Although various methods can be used to detect these apple-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 apple-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 apple proteins.

[0025] The type of test sample to be used in the apple 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 apples at a food ingredient production factory that handles apples. 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 apples at a food production factory that handles apples. Furthermore, when producing processed foods that do not contain apples after producing processed foods that contain apples, careful cleaning of the food production equipment is essential, with the removal of apple residues in mind. To verify the effectiveness of this cleaning method and to confirm the presence or absence of apple residues in the food production equipment, wiped samples from the production equipment are also examples of 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 apple protein standard samples with known concentrations To verify the quantitative capability of the apple detection method by LC-MS / MS of the present invention, standard samples with known apple protein concentrations were analyzed and a calibration curve was created.

[0028] Proteins were extracted from apples 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] After adding 10 μL of a bovine pancreas trypsin solution adjusted to 20 mg / mL with 0.1% formic acid, the sample was left to stand overnight at 37°C to perform enzymatic digestion of the apple 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 in the sample of 1.25 - 20 μg / mL in terms of total apple protein was prepared and analyzed by LC-MS / MS.

[0035] <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 [[ID=2i7]]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 apple protein-derived peptide fragments targeted for detection are shown in Table 1. [[ID=X]]

[0037]

Table 1

[0038] FIG. 1 shows an example of a chromatogram obtained when a standard sample with a total apple protein concentration of 1.25 μg / mL was analyzed (peptide sequence: LVASGSGSIIK (SEQ ID NO: 1), Q1: 516.3, Q3: 819.5).

[0039] Figure 2 shows an example of a calibration curve under the same detection conditions as Figure 1. A good calibration curve with R2: 0.998 was obtained in the range of 1.25 to 20 ppm, calculated as the concentration of apple total protein in the sample.

[0040] Example 2 Apple protein spike test for processed foods To examine the applicability of the apple detection method by LC-MS / MS of the present invention to processed foods, an apple-free white rice porridge was analyzed after adding an apple protein standard sample to a concentration of 10 ppm.

[0041] 1 g of a white rice porridge sample not containing apple was weighed into a 50 mL polypropylene centrifuge tube, and the apple protein standard sample used in Example 1 was added to give a total apple 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] The chromatogram of the white rice porridge sample containing no apple is shown in Figure 3, and the chromatogram of the sample to which an apple protein standard sample was added at 10 ppm is shown in Figure 4 (peptide sequence: LVASGSGSIIK (SEQ ID NO: 1), Q1: 516.3, Q3: 819.5).

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

Claims

1. A method for detecting apples, comprising the steps of extracting proteins from a sample, treating the extracted proteins with a protease to obtain enzymatic digests, analyzing the enzymatic digests, and detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 3 using a mass spectrometer to qualitatively or quantitatively determine whether or not apple proteins are 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 516 / 820, 516 / 748, 516 / 213, or 516 / 661 ii) SEQ ID NO: 2, m / z value of about 476 / 606 iii) SEQ ID NO: 3, m / z values ​​of about 528 / 897, 528 / 737, 528 / 449, 528 / 624, 528 / 553, 528 / 234, 518 / 319, or 518 / 406 and a step of qualitatively or quantitatively determining whether or not an apple 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 an apple 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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