Squid detection method using a mass spectrometer

Abstract

To enable high-sensitivity detection of squid that might cause allergies contained in food ingredients and products even if the amount is minute.SOLUTION: A method of detecting squid 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 squid protein in the sample.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a detection method using a mass spectrometer that enables highly sensitive detection of even trace amounts of any food raw materials, products, etc. that may cause food allergies.

Background Art

[0002] Decapodiformes are animals of the superorder Decapodiformes in the phylum Mollusca, and in Japan, they are designated as "substances conforming to specific raw materials" for which labeling is recommended as substances that may cause food allergies (Food Labeling Standards, March 30, 2015, Food Labeling Notice No. 139).

[0003] Foods that may cause allergies can be accidentally contaminated with trace amounts during production, distribution, and processing. Therefore, as providers of food raw materials or products, it is important to conduct quality control to check whether they are contaminated.

[0004] As methods for inspecting the presence or absence of contamination with specific foods, there are methods for detecting characteristic proteins using antigen-antibody reactions such as the ELISA method, Western blot method, and immunochromatography method, and methods for detecting characteristic DNA base sequences by the PCR method.

[0005] In recent years, a method for detecting peptides derived from proteins characteristic of specific foods using a mass spectrometer has been reported. This is a technique that can quantify the proteins of the target raw materials, and has the advantages of reducing false positive reactions that are likely to occur when using antigen-antibody reactions, and enabling simultaneous detection of multiple items.

[0006] As prior art related to the detection of Decapodiformes, for example, the following prior art is disclosed.

Prior Art Documents

Patent Documents

[0007] [Patent Document 1] Patent No. 4436438 [Non-patent literature]

[0008] [Non-Patent Document 1] 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 in question focus on detecting genes, and other methods may also be possible. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] Therefore, the 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, from food ingredients and products. [Means for solving the problem]

[0011] To achieve the above objectives, the inventors focused on the amino acid sequence of the squid protein to be detected and diligently researched methods for specifically and highly sensitively detecting squid. As a result, they discovered characteristic amino acid sequences of squid and found that by detecting these amino acid sequences, squid can be detected specifically and highly sensitively, thus completing the present invention. Specifically, the present invention relates first to the following items.

[0012] Section 1. A detection method comprising the steps of extracting a protein from a sample, obtaining an enzymatic digest of the extracted protein using a proteolytic enzyme, and analyzing the enzymatic digest to qualitatively or quantitatively determine whether or not a protein is present in the sample by detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 2 using a mass spectrometer.

[0013] Next, as a method for detecting at least one peptide selected from the group consisting of Sequence IDs 1 and 2 above, it is preferable to analyze 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 item 2 below.

[0014] Section 2. The process involves extracting proteins from a sample, obtaining an enzymatic digest of the extracted proteins using proteolytic enzymes, and analyzing the enzymatic digest by liquid chromatography-tandem mass spectrometry (LC-MS / MS), as follows: i) m / z values ​​for sequence number 1, approximately 488 / 676, 488 / 300, 488 / 605, and 488 / 789 ii) M / Z values ​​of approximately 551 / 760, 551 / 890, 551 / 989, 551 / 647, 1102 / 342, 1102 / 213, 1102 / 842, or 1102 / 518, according to Sequence ID 2. A method for detecting squid, comprising the step of determining whether or not squid 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 the following.

[0015] Next, it is preferable to monitor two or more of the precursor-product ion pair transitions. That is, the present invention relates to item 3 below. Section 3. A step of qualitatively or quantitatively determining whether any protein is present in a sample by monitoring at least two or more precursor-product ion pair transitions having specific m / z values related to the specific amino acid sequence, and the detection method according to claim 2 including the step.

Advantages of the Invention

[0016] According to the present invention, it is possible to detect peptides derived from any protein by LC-MS / MS analysis, and it has the effect of enabling quality control inspections such as whether any of the above is mixed in or used in a test food raw material or a test food. It can also contribute to preventing allergies in advance and investigating causative substances when allergic symptoms occur.

Brief Description of the Drawings

[0017] [Figure 1] Peaks of peptides derived from any protein in a chromatogram obtained from a standard sample with a known concentration of any [Figure 2] A calibration curve created by plotting the area of peptides derived from any protein in a chromatogram obtained from a standard sample with a known concentration of any and the known concentration of any protein in the standard sample [Figure 3] An exemplary chromatogram of a white miso sample not containing any [Figure 4] An exemplary chromatogram containing peptides derived from any protein obtained from a white miso sample spiked with any protein

Modes for Carrying Out the Invention

[0018] The present invention provides a method for detecting trace amounts of protein present in test samples such as food ingredients and processed foods. Specifically, the method includes the steps of: extracting protein from the test sample; obtaining an enzymatic digest of the extracted protein using a proteolytic enzyme; and analyzing the enzymatic digest by LC-MS / MS to obtain a chromatogram of the target peptide. Preferred embodiments of the method according to this embodiment will be described below.

[0019] For extracting proteins from the test sample, buffer solutions containing surfactants or commercially available protein analysis kits can be used.

[0020] It is preferable to further reduce and alkylate the protein extract from the test sample to block the thiol groups.

[0021] The sample prepared as described above is treated with a proteolytic enzyme. Examples of proteolytic enzymes used in the method of the present invention include trypsin and chymotrypsin, with trypsin being preferred. The treatment conditions can be appropriately selected depending on the type of enzyme. This enzymatic treatment degrades the target protein and generates multiple peptides.

[0022] The obtained enzyme digest should preferably be purified by removing surfactants and using a reversed-phase solid-phase column before being analyzed by LC-MS / MS.

[0023] The peptide sequences analyzed by LC-MS / MS are as follows: Sequence ID 1 WIAEDADR Sequence ID 2 IVELEEELK Various methods can be used to detect these squid-derived peptides, but in this invention, a mass spectrometer is used. Of these, methods utilizing liquid chromatography are particularly preferred. For example, methods using LC-MS or LC-MS / MS are available. In particular, it is preferable to remove surfactants from the obtained enzyme digest and purify it using a reversed-phase solid-phase column before analyzing it by LC-MS / MS.

[0024] Furthermore, it is possible to perform quantitative analysis of squid protein by processing standard samples with known squid protein concentrations in the same way as the test samples, analyzing them by LC-MS / MS, and creating a calibration curve.

[0025] In the squid detection method of the present invention, the type of test sample is not particularly limited. For example, test samples include food ingredients and processed foods. Food ingredients include food ingredients that are intentionally produced separately from squid at food ingredient production plants that handle squid. Processed foods include confectionery, noodles, powdered soups, liquid soups, hot-air dried or freeze-dried ingredients, or various cooked foods containing these processed foods. Also, processed foods that are intentionally produced separately from squid at food manufacturing plants that handle squid are also included. Furthermore, when manufacturing processed foods that do not contain squid after manufacturing processed foods that contain squid, thorough cleaning of the food manufacturing equipment with the removal of squid residue in mind is essential. From the viewpoint of confirming the effectiveness of this cleaning method and the presence or absence of squid residue in the food manufacturing equipment, wipe samples from the manufacturing equipment can also be used as test samples.

[0026] Examples The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Furthermore, it is possible to modify the present invention as appropriate without departing from its spirit.

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

[0028] Depending on whether you want to purchase from a store, proteins were extracted using MPEX PTS Reagents (60 mM SDC SLS / 50 mM TEAB) (manufactured by GL Sciences Inc.), and the total protein concentration was determined using a 2-D Quant Kit (manufactured by Cytiva) to serve as a standard sample.

[0029] Among the prepared standard samples, 40 μg of protein was collected in a 2.0 mL low-adsorption polypropylene tube, 1000 μg of egg-derived ovalbumin and 100 μg of bovine-derived albumin were added, and the total solution volume was made 700 μL.

[0030] 70 μL of 1M TEAB and 28 μL of 1M DTT were added, and after standing at 75°C for 15 minutes and then at room temperature for 30 minutes. Next, 56 μL of an iodoacetamide solution prepared to 1M with distilled water was added, and after standing at room temperature in the dark for 45 minutes, 28 μL of 1M DTT was added (reduction and alkylation).

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

[0032] Formic acid was added to the obtained enzymatic digest to make it acidic, and then ethyl acetate was added to remove the surfactant contained in the extraction solution by liquid-liquid partitioning. The removal operation 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 in the sample of total protein converted to 1.25 - 20 μg / mL was prepared and analyzed by LC-MS / MS.

[0035] <LC-MS / MS instrument> 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 peptide fragments derived from the target protein to be detected are shown in Table 1.

[0037]

Table 1

[0038] The chromatogram when analyzing a standard sample with a total protein concentration of 1.25 μg / mL is illustrated in Figure 1 (peptide sequence: WIAEDADR (SEQ ID NO: 1), Q1: 488.2, Q3: 676.3).

[0039] The calibration curve under the same detection conditions as in Figure 1 is illustrated in Figure 2. A good calibration curve with R2: 0.984 was obtained in the range of 从1.25 to 20 ppm in terms of the converted value of the concentration of the total protein in the sample.

[0040] Example 2 Squid protein spike testing in processed foods To investigate the applicability of the LC-MS / MS squid detection method of the present invention to processed foods, a sample of squid protein standard was added to white rice porridge without squid to a concentration of 10 ppm and then analyzed.

[0041] One g of a white rice porridge sample without squid was weighed into a 50 mL polypropylene centrifuge tube, and the squid protein standard sample used in Example 1 was added to achieve a total squid protein concentration of 10 ppm.

[0042] 30 μL of ethylenediaminetetraacetic acid (EDTA), prepared to a concentration of 100 mg / mL in a 1N sodium hydroxide solution, was added.

[0043] 9 mL of the extraction solution used in Example 1 was added, and the mixture was shaken overnight at 90-110 rpm to extract the protein.

[0044] The mixture was centrifuged at 4°C and 10,000xg for 30 minutes, and 700 μL of the supernatant was collected in a 2.0 mL low-adsorption polypropylene tube.

[0045] The subsequent steps were carried out in the same manner as in Example 1, and the final dissolved solution was analyzed by LC-MS / MS.

[0046] Figure 3 shows a chromatogram of a plain rice porridge sample without squid, and Figure 4 shows a chromatogram of a sample to which a standard squid protein sample was added at a concentration of 10 ppm (peptide sequence: WIAEDADR (SEQ ID NO: 1), Q1:488.2, Q3:676.3).

[0047] The target peak was only detected when a standard sample of squid protein was added.

Claims

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

2. The process involves extracting proteins from a sample, treating the extracted proteins with proteolytic enzymes to obtain an enzymatic digest, and analyzing the enzymatic digest by liquid chromatography-tandem mass spectrometry (LC-MS / MS), as follows: i) m / z values ​​for sequence number 1, approximately 488 / 676, 488 / 300, 488 / 605, and 488 / 789 ii) m / z values ​​of sequence number 2, approximately 551 / 760, 551 / 890, 551 / 989, 551 / 647, 1102 / 342, 1102 / 213, 1102 / 842, or 1102 / 518 A method for detecting squid, comprising the step of qualitatively or quantitatively determining whether or not squid 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 the following.

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

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