Method for detecting squid using mass spectrometer

Abstract

[Problem] The present invention performs high-sensitivity detection of squid, which can cause an allergic reaction, even when the squid is contained in a very small amount in a food raw material, a product, etc. [Solution] Provided is a method for detecting squid, the method comprising a step for extracting a protein from a sample, a step for 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 and 2 using a mass spectrometer to qualitatively or quantitatively determine whether a squid protein is present in the sample.

Description

Method for detecting squid using a mass spectrometer

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

[0002] Squid (Decapodiformes) are animals of the phylum Mollusca and the superorder Decapoda. In Japan, they are 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 such detection, for example, the following prior art has been disclosed.

[0007] Patent No. 4436438

[0008] Amaya Velasco, Graciela Ramilo-Fernandez, Carmen G Sotelo, A Real-Time PCR Method for the Authentication of Common Cuttlefish (Sepia officinalis) in Food Products; Foods., 9(3): 286, 2020.

[0009] On the other hand, the patent and non-patent documents are concerned with detecting genes, and other methods may also be possible.

[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 squid, which may cause allergies, in food raw materials and products.

[0011] In order to achieve the above object, the present inventors have focused on the amino acid sequences of the squid protein to be detected and have intensively studied a method for specifically and highly sensitively detecting squid. As a result, they have found amino acid sequences characteristic of squid and found that detecting these amino acid sequences enables specific and highly sensitive detection of squid, which has led to the completion of the present invention. That is, the present invention first relates to the following items.

[0012] Item 1. A method for detecting protein, 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 and 2 using a mass spectrometer to qualitatively or quantitatively determine whether or not protein is present in the sample.

[0013] Next, a preferred method for detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 and 2 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.

[0014] Item 2. A method for detecting squid, comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein with a protease, and analyzing the enzymatic digest by liquid chromatography tandem mass spectrometry (LC-MS / MS) to determine whether or not squid protein is 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 488 / 676, 488 / 300, 488 / 605, and 488 / 789; and ii) SEQ ID NO: 2, m / z values ​​of approximately 551 / 760, 551 / 890, 551 / 989, 551 / 647, 1102 / 342, 1102 / 213, 1102 / 842, or 1102 / 518.

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

[0016] The present invention enables the detection of squid protein-derived peptides by LC-MS / MS analysis, thereby enabling quality control testing to determine whether or not the above-mentioned squid is mixed into or used in test food ingredients or test foods. It can also contribute to the prevention of allergies and the investigation of causative substances when allergic symptoms occur.

[0017] Peaks of squid protein-derived peptides in a chromatogram obtained from a standard sample with a known squid concentration. A calibration curve created by plotting the area of ​​squid protein-derived peptides in a chromatogram obtained from a standard sample with a known squid concentration against the known squid protein concentration in the standard sample. An exemplary chromatogram of a squid-free rice porridge sample. An exemplary chromatogram containing squid protein-derived peptides obtained from a rice porridge sample spiked with squid protein.

[0018] The present invention provides a method for detecting trace amounts of contaminating proteins in test samples, such as food raw materials and processed foods. Specifically, the method includes the steps of extracting proteins from the test sample, obtaining enzymatic digests of the extracted proteins with protease, and analyzing the enzymatic digests by LC-MS / MS to obtain chromatograms 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 WIAEDADR SEQ ID NO: 2 IVELEEELK Although various methods can be used to detect these squid-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.

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

[0025] The type of test sample to be used in the squid 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 containing squid in a food ingredient production factory that handles squid. 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 containing squid in a food production factory that handles squid. Furthermore, when producing processed foods that do not contain squid after producing processed foods containing squid, careful cleaning of the food production equipment is essential, with the removal of squid residue in mind. To verify the effectiveness of this cleaning method and the presence or absence of squid residue in the food production equipment, wiped samples from the production equipment can also be used as test samples.

[0026] 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.

[0027] Example 1 Analysis of Standard Samples with Known Squid Protein Concentrations To verify the quantitative capability of the squid detection method by LC-MS / MS of the present invention, standard samples with known squid protein concentrations were analyzed and a calibration curve was created.

[0028] Proteins were extracted using MPEX PTS Reagents (60 mM SDC SLS / 50 mM TEAB) (GL Sciences) purchased from a store, 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, 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).

[0031] After adding 10 μL of a trypsin solution derived from bovine pancreas adjusted to 20 mg / mL with 0.1% formic acid, the sample was left to stand overnight at 37°C for enzymatic digestion of the squid 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 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.

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

[0035] <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

[0036] The sequences and MRM transitions of the peptide fragments derived from squid proteins to be detected are shown in Table 1.

[0037]

[0038] The chromatogram obtained by analyzing a standard sample containing 1.25 μg / mL of squid total protein is shown in FIG. 1 (peptide sequence: WIAEDADR (SEQ ID NO: 1), Q1: 488.2, Q3: 676.3).

[0039] Figure 2 shows an example of a calibration curve under the same detection conditions as Figure 1. A good calibration curve with R2: 0.984 was obtained in the range of 1.25 to 20 ppm, calculated as the total squid protein concentration in the sample.

[0040] Example 2: Squid protein spiking test in processed foods To examine the applicability of the squid detection method of the present invention using LC-MS / MS to processed foods, squid-free white rice porridge was spiked with a standard sample of squid protein at 10 ppm and analyzed.

[0041] 1 g of squid-free white rice porridge sample was weighed into a 50 mL polypropylene centrifuge tube, and the squid protein standard sample used in Example 1 was added to give a total squid 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 squid-free white rice porridge sample is shown in Figure 3, and the chromatogram of the sample to which a squid protein standard sample was added at 10 ppm is shown in Figure 4 (peptide sequence: WIAEDADR (SEQ ID NO: 1), Q1: 488.2, Q3: 676.3).

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

[0048] This application is based on Japanese application No. 2024-045715 filed on March 21, 2024 and Japanese application No. 2024-092598 filed on June 6, 2024, the contents of which are incorporated herein by reference.

Claims

1. A method for detecting protein, 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 protein is present in the sample.

2. A method for detecting squid, 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 or not squid 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) SEQ ID NO: 1, m / z values ​​of about 488 / 676, 488 / 300, 488 / 605, and 488 / 789; and ii) SEQ ID NO: 2, m / z values ​​of about 551 / 760, 551 / 890, 551 / 989, 551 / 647, 1102 / 342, 1102 / 213, 1102 / 842, or 1102 / 518.

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