Method for detecting soybean using mass spectrometer

Abstract

[Problem] To detect soybean, which has the potential to cause an allergic reaction, in a food ingredient, a product, or the like, at high sensitivity even when the content thereof is extremely small. [Solution] Provided is a method for detecting soybean whereby the presence or absence of soy proteins in a sample can be qualitatively or quantitatively determined by carrying out a step for extracting a protein from the sample and a step for obtaining an enzymatic digest from the extracted protein through use of a proteolytic enzyme, analyzing the enzymatic digest, and using a mass spectrometer to detect at least one peptide selected from the group consisting of SEQ ID NOs: 1-3.

Description

Soybean detection method using a mass spectrometer

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

[0002] Soybeans (Glycine max) are a plant of the genus Glycine in the family Fabaceae, and 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 soybeans, for example, the following prior art by the applicant has been disclosed.

[0007] Patent No. 4937306

[0008] On the other hand, the patent document is directed to detecting genes, and other methods are possible.

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

[0010] In order to achieve the above object, the present inventors have focused on the amino acid sequences of the soybean allergen proteins to be detected and have intensively studied methods for specifically and highly sensitively detecting soybeans. As a result, they have found amino acid sequences characteristic of soybeans, and have discovered that detecting these amino acid sequences enables specific and highly sensitive detection of soybeans, thereby completing the present invention. That is, the present invention first relates to the following items.

[0011] Item 1. A soybean detection method comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein with a protease, analyzing the enzymatic digest, 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 soybean protein is present in the sample.

[0012] Next, a preferred method for detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 3 is to analyze the peptide 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.

[0013] Item 2. A method for detecting soybeans, comprising the steps of extracting proteins from a sample, obtaining an enzymatic digest of the extracted proteins with a protease, and analyzing the enzymatic digest by liquid chromatography tandem mass spectrometry (LC-MS / MS) to determine whether or not soybean proteins are present in the sample by monitoring at least one or more precursor-product ion pair transitions having specific m / z values ​​associated with specific amino acid sequences selected from the group consisting of: i) SEQ ID NO: 1, m / z values ​​of approximately 484 / 381, 484 / 643, or 484 / 294; ii) SEQ ID NO: 2, m / z values ​​of approximately 713 / 213, 713 / 326, 713 / 515, 476 / 416, 476 / 515, or 476 / 213; and iii) SEQ ID NO: 3, m / z values ​​of approximately 530 / 311, 530 / 147, or 530 / 424.

[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. Item 3. The soybean detection method according to claim 2, which comprises a step of qualitatively or quantitatively determining whether or not soybean 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.

[0015] According to the present invention, it is possible to detect peptides derived from soy protein by LC-MS / MS analysis, thereby enabling quality control inspections to be carried out to determine whether or not the above-mentioned soybeans are mixed into 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.

[0016] Peaks of soy protein-derived peptides in a chromatogram obtained from a standard sample with a known soybean concentration. Calibration curve created by plotting the area of ​​soy protein-derived peptides in a chromatogram obtained from a standard sample with a known soybean concentration against the known soybean protein concentration in the standard sample. Exemplary chromatogram of an instant noodle sample that does not contain soybeans. Exemplary chromatogram containing soy protein-derived peptides obtained from an instant noodle sample spiked with soy protein.

[0017] The present invention provides a method for detecting trace amounts of soybean protein present in a test sample, such as a food material or processed food. 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 SQSDNFEYVSFK SEQ ID NO: 2 VLIVPQNFVVAAR SEQ ID NO: 3 FYLAGNQEQEFLK Although various methods can be used to detect these soybean-derived peptides, the present invention utilizes a mass spectrometer. Among these, methods using liquid chromatography are particularly preferred. Examples include methods using LC-MS and LC-MS / MS. In particular, it is preferable to analyze the obtained enzymatic digests by LC-MS / MS after removing the surfactant and purifying them using a reverse-phase solid-phase column.

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

[0024] The type of test sample to be used in the soybean 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 soybeans at a food ingredient production factory that handles soybeans. 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 soybeans at a food production factory that handles soybeans. Furthermore, when producing processed foods that do not contain soybeans after producing processed foods that contain soybeans, careful cleaning of the food production equipment is essential, with the removal of soybean residues in mind. From the perspective of verifying the effectiveness of this cleaning method and the presence or absence of soybean residues in the food production equipment, wiped samples from the production equipment can also be used as test samples.

[0025] 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, appropriate modifications can be made without departing from the gist of the present invention.

[0026] Example 1 Analysis of Standard Samples with Known Soybean Protein Concentrations To verify the quantitative capability of the soybean detection method by LC-MS / MS of the present invention, standard samples with known soybean protein concentrations were analyzed and a calibration curve was created.

[0027] Proteins were extracted from soybeans 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 the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) to serve as a standard sample.

[0028] Of the prepared standard sample, 50 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, followed by 30 minutes at room temperature. 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 and alkylation).

[0030] 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 soybean 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 purified solution was dried using a centrifugal evaporator, dissolved in 5% acetonitrile with 0.1% formic acid, and diluted to a total soy protein concentration of 1.25 to 10 μg / mL to prepare a dilution series, which was then analyzed by LC-MS / MS.

[0034] <LC-MS / MS device> LC section: ExionLC AD system (SCIEX) MS / MS section: QTRAP (registered trademark) 6500+ system (SCIEX) <LC conditions> Analytical 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 analysis conditions> Ionization: Electrospray ionization Polarity: Positive Spray voltage: 5500 V

[0035] The sequences and MRM transitions of the peptide fragments derived from soybean proteins that were the targets of detection are shown in Table 1.

[0036]

[0037] FIG. 1 shows an example of a chromatogram obtained by analyzing a standard sample with a soybean total protein concentration of 1.25 μg / mL (peptide sequence: VLIVPQNFVVAAR (SEQ ID NO: 2), Q1: 713.4, Q3: 213.2).

[0038] Figure 2 shows an example of a calibration curve under the same detection conditions as Figure 1. A good calibration curve with R2: 0.999 was obtained in the soybean total protein concentration range of 1.25 to 10 ppm.

[0039] Example 2: Soy protein spiking test in processed foods In order to examine the applicability of the soy detection method by LC-MS / MS of the present invention to processed foods, a soy protein standard sample was added to soybean-free instant noodle powder to a concentration of 10 ppm, and then analyzed.

[0040] 1 g of a sample of ground instant noodles that does not contain soybeans was weighed into a 50 mL polypropylene centrifuge tube, and the soybean protein standard sample used in Example 1 was added to give a total soybean 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] The chromatogram of a sample of ground instant noodles that does not contain soybeans is shown in Figure 3, and the chromatogram of a sample to which a soy protein standard sample was added to make the product content 10 ppm is shown in Figure 4 (peptide sequence: VLIVPQNFVVAAR (SEQ ID NO: 2), Q1: 713.4, Q3: 213.2).

[0046] The target peak was observed only when the soy protein standard sample was added.

Claims

1. A method for detecting soybeans, 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 to 3 using a mass spectrometer to qualitatively or quantitatively determine whether or not soybean protein is present in the sample.

2. A method for detecting soybean, comprising the steps of extracting proteins from a sample, treating the extracted proteins with a protease to obtain an enzymatic digest, and analyzing the enzymatic digest by liquid chromatography tandem mass spectrometry (LC-MS / MS) to qualitatively or quantitatively determine whether soybean protein is present in the 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: i) m / z values ​​of about 484 / 381, 484 / 643, or 484 / 294 for SEQ ID NO:1; ii) m / z values ​​of about 713 / 213, 713 / 326, 713 / 515, 476 / 416, 476 / 515, or 476 / 213 for SEQ ID NO:2; and iii) m / z values ​​of about 530 / 311, 530 / 147, or 530 / 424 for SEQ ID NO:

3.

3. The method for detecting soybeans according to claim 2, further comprising a step of qualitatively or quantitatively determining whether or not soybean protein is 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.

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