JP7837366B2 - Squid detection method using a mass spectrometer - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law (1) Abstracts of the lectures Abstracts of the 29th General Meeting and Scientific Conference of the Japan Society for Food Chemistry A11. Development of a simultaneous quantitative testing method for 20 recommended labeling items using LC-MSMS (2) Presentation at the conference Lecture A11 of the 29th General Meeting and Scientific Conference of the Japan Society for Food Chemistry. Development of a simultaneous quantitative testing method for 20 recommended labeling items using LC-MSMS (3) Abstracts of the lectures Abstracts of the 119th Scientific Conference of the Japan Society for Food Hygiene P-68: Development of a simultaneous quantitative testing method for 20 recommended food allergens, Part 2: Examination of the scope of application to biological species, varieties, etc. (4) Presentation at the conference Lecture P-68 of the 119th Scientific Conference of the Japan Society for Food Hygiene: Development of a simultaneous quantitative testing method for 20 recommended food allergens, Part 2: Examination of the scope of application to biological species, varieties, etc.

This invention relates to a method for detecting squid using a mass spectrometer, which enables highly sensitive detection of squid, even in trace amounts, when it is present in food ingredients or products that may cause food allergies.

Squid (Decapodiformes) is an animal belonging to the phylum Mollusca, superorder Decapodiformes, and in Japan, it is designated as a "substance similar to a specified allergen," for which labeling is recommended due to its potential to cause food allergies (Regarding Food Labeling Standards, March 30, 2015, Consumer Affairs Agency Notification No. 139).

Because unintentional contamination of food ingredients or products with potential allergens can occur during production, distribution, and processing, it is crucial for providers of food ingredients or products to implement quality control measures to ensure that no such contamination is present.

Methods for testing for the presence of specific food contaminants include methods that use antigen-antibody reactions such as ELISA, Western blotting, and immunochromatography to detect characteristic proteins, as well as methods that detect characteristic DNA base sequences using PCR.

Furthermore, in recent years, methods have been reported for detecting peptides derived from proteins characteristic of specific foods using mass spectrometry. This technique allows for the quantitative analysis of the target raw material and has the advantages of reducing false positive reactions that are common with antigen-antibody reactions, as well as enabling the simultaneous detection of multiple items.

The following prior art has been disclosed as prior art related to squid detection:

Patent No. 4436438

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.

On the other hand, the patent and non-patent documents in question focus on detecting genes, and other methods may also be possible.

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.

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.

Item 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.

Next, as a method for detecting at least one peptide selected from the group consisting of Sequence IDs 1 and 2 above, a preferred method is 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.

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) 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 m/z values of approximately 551/760, 551/890, 551/989, 551/647, 1102/342, 1102/213, 1102/842, or 1102/518, as shown in SEQ ID NO: 2.

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 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.

According to the present invention, LC-MS/MS analysis enables the detection of peptides derived from squid protein, allowing for quality control inspections to determine whether or not the above-mentioned squid is present in or used in the raw materials or test food products. Furthermore, it can contribute to the prevention of allergies and the investigation of causative substances when allergic symptoms occur.

Peak of squid protein-derived peptide in chromatogram obtained from standard samples with known squid concentration. A calibration curve was created by plotting the area of squid protein-derived peptides in chromatograms obtained from standard samples with known squid concentrations against the known squid protein concentrations in those standard samples. Exemplary chromatogram of a white rice porridge sample without squid. Exemplary chromatogram containing squid protein-derived peptides obtained from a white rice porridge sample spiked with squid protein.

The present invention provides a method for detecting trace amounts of trace proteins in test samples such as food ingredients and processed foods. Specifically, the method comprises the steps of: extracting proteins from the test sample; obtaining an enzymatic digest of the extracted proteins 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.

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

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

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, generating multiple peptides.

The obtained enzyme digest should preferably be analyzed by LC-MS/MS after removing surfactants and purifying it using a reversed-phase solid-phase column.

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.

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

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.

Examples <br/> 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 invention as appropriate without departing from the spirit of the invention.

Example 1
Analysis of standard samples with known squid protein concentrations <br/> 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.

Proteins were extracted from squid purchased from a store using MPEX PTS Reagents (60 mM SDC SLS/50 mM TEAB) (GL Sciences Co., Ltd.), and the total protein concentration was determined using the 2-D Quant Kit (Cytiva Co., Ltd.) to create the standard sample.

From the prepared standard sample, 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, bringing the total solution volume to 700 μL.

70 μL of 1M TEAB and 28 μL of 1M DTT were added, and the mixture was allowed to stand at 75°C for 15 minutes, followed by 30 minutes at room temperature. Next, 56 μL of iodoacetamide solution prepared to 1M with distilled water was added, and the mixture was allowed to stand at room temperature under light protection for 45 minutes, after which 28 μL of 1M DTT was added (reduction and alkylation).

A 10 μL solution of bovine pancreas-derived trypsin, prepared to 20 mg/mL with 0.1% formic acid, was added, and the mixture was allowed to stand overnight at 37°C before enzymatic digestion of the squid standard sample.

Formic acid was added to the obtained enzyme digest to create an acidic state, and then ethyl acetate was added to remove the surfactant contained in the extract by liquid-liquid partitioning. This removal procedure was repeated three times.

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

The purified solution was dried using a centrifugal evaporator and dissolved in 0.1% formic acid containing 5% acetonitrile. A dilution series ranging from 1.25 to 20 μg/mL (converted to total squid protein in the sample) was prepared and analyzed by LC-MS/MS.

<LC-MS/MS equipment>
LC section: ExionLC ADsystem (SCIEX Corporation)
MS/MS Department: QTRAP(registered trademark) 6500+ system (SCIEX Corporation)
<LC conditions>
Analytical column: YMC-Triart C18, particle size 3 μm, 100 x 2.1 mm id. (YMC Corporation)
Column temperature: 40°C
Column flow rate: 0.3 mL/min
Eluent A: 0.1% formic acid; Eluent B: 0.1% formic acid-containing acetonitrile gradient. 0 min (B: 5%) → 16 min (B: 40%) → 18 min (B: 95%) → 23 min (B: 95%) → 23.1 min (B: 5%) → Reset <Mass spectrometry conditions>
Ionization: Electrospray ionization method Polarity: Positive spray Voltage: 5500 V

Table 1 shows the sequences and MRM transitions of the peptide fragments derived from the squid protein that were targeted for detection.

Figure 1 shows an example of a chromatogram obtained by analyzing a standard sample of squid with a total protein concentration of 1.25 μg/mL (peptide sequence: WIAEDADR (SEQ ID NO: 1), Q1: 488.2, Q3: 676.3).

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

Example 2
Squid Protein Spike Test in Processed Foods <br/> To investigate the applicability of the LC-MS/MS squid detection method of the present invention to processed foods, a squid protein standard sample was added to white rice porridge without squid to a concentration of 10 ppm and analyzed.

One g of 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.

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

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.

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.

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

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

The target peak was only detected when a standard squid protein sample was added.

Claims (3)

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.
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) A method for detecting the following protein, comprising the step of qualitatively or quantitatively determining whether the following 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 m/z values of approximately 551/760, 551/890, 551/989, 551/647, 1102/342, 1102/213, 1102/842, or 1102/518, as shown in SEQ ID NO: 2.
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.