Method for detecting salmon using mass spectroscope

Abstract

To detect a salmon potentially causing an allergic reaction even with a trace quantity thereof included in a raw material of a food product or manufactured articles at high sensitivity.SOLUTION: A method for detecting a salmon performs: a step of extracting a protein from a specimen; a step of obtaining an enzymatic digestion product from the extracted protein by a protease; and analyzing the enzymatic digestion product and detecting one or more peptides selected from a group consisting of sequence numbers 1 to 2, using a mass spectroscope. The method thus determines whether or not a salmon protein is present in the specimen qualitatively or quantitatively.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for detecting salmon using a mass spectrometer, which enables high sensitivity detection of trace amounts of salmon, which may cause food allergies, when it is contained in food ingredients or products. [Background technology]

[0002] Salmon (Oncorhynchus keta, etc.) is a fish of the Salmonidae family, Salmon genus, and in Japan, it is designated as a "substance equivalent to a specific raw material" that is recommended to be labeled as a substance that can cause food allergies (Food Labeling Standards, Food and Nutrition Information Table Notification No. 139, 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 carry out quality control to check whether or not such substances are present.

[0004] Methods for testing for the presence or absence of contamination of specific foods include methods that use antigen-antibody reactions such as ELISA, Western blot, and immunochromatography to detect characteristic proteins, and methods that use PCR to detect characteristic DNA base sequences.

[0005] Recently, a method has been reported that uses a mass spectrometer to detect peptides derived from proteins that are characteristic of certain foods. This technology can quantify the protein in the target raw materials, and has the advantages of reducing false positive reactions that tend to occur when using antigen-antibody reactions, and of being able to detect multiple items simultaneously.

[0006] As prior art relating to salmon detection, for example, the following prior art by the applicant has been disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 4439426

[0008] On the other hand, the patent document is directed to detection of genes, and other methods are possible. Summary of the Invention [Problem to be solved by the invention]

[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 salmon, which may cause allergies, in food raw materials and products. [Means for solving the problem]

[0010] In order to achieve the above object, the present inventors have focused on the amino acid sequences in the salmon allergen protein to be detected and have intensively studied a method for specifically and highly sensitively detecting salmon. As a result, they have found amino acid sequences characteristic of salmon, and have found that detecting these amino acid sequences makes it possible to specifically and highly sensitively detect salmon, thereby completing the present invention. That is, the present invention first relates to the following items.

[0011] Section 1. A method for detecting salmon, comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein using a proteolytic enzyme, analyzing the enzymatic digest, and detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 4 using a mass spectrometer to qualitatively or quantitatively determine whether or not salmon protein is present in the sample.

[0012] Next, as a method for detecting at least one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 2, a method is preferably used in which at least one or more precursor-product ion pair transitions having specific m / z values ​​associated with specific amino acid sequences are monitored by liquid chromatography tandem mass spectrometry (LC-MS / MS). That is, the present invention relates to the following item 2.

[0013] Section 2. A step of extracting proteins from a sample, a step of obtaining an enzymatic digest of the extracted proteins with 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 638 / 342, 638 / 820, 426 / 577, or 426 / 289 ii) SEQ ID NO:2, m / z values ​​of about 702 / 1107, 702 / 975, or 702 / 846 and determining whether or not salmon 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:

[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. Section 3. and determining qualitatively or quantitatively whether or not salmon 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. Effect of the Invention

[0015] According to the present invention, it is possible to detect peptides derived from salmon proteins by LC-MS / MS analysis, and it is possible to carry out quality control inspections to determine whether or not the above-mentioned salmon is mixed into or used in test food ingredients or test foods. It can also contribute to preventing allergies and investigating causative substances when allergic symptoms occur. [Brief description of the drawings]

[0016] [Figure 1] Salmon protein peptide peaks in chromatograms obtained from standard samples with known salmon concentrations [Diagram 2] A calibration curve was prepared by plotting the area of ​​peptides derived from salmon protein in a chromatogram obtained from a standard sample with a known salmon concentration against the known salmon protein concentration in the standard sample. [Diagram 3] An exemplary chromatogram of a white rice porridge sample that does not contain salmon [Figure 4] An exemplary chromatogram containing salmon protein-derived peptides obtained from a white rice porridge sample spiked with salmon protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention provides a method for detecting salmon proteins present in trace amounts in test samples such as food raw materials and processed foods. That is, 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 peptides to be analyzed. A preferred embodiment of the method according to this embodiment will be 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 the protease used in the method of the present invention include trypsin and chymotrypsin, and trypsin is preferred. The treatment conditions may be appropriately selected depending on the type of enzyme. The enzyme treatment degrades the target protein to generate multiple peptides.

[0021] It is desirable to analyze the obtained enzymatic digests by LC-MS / MS after removing the surfactant and purifying them using a reverse phase solid phase column.

[0022] The peptide sequences analyzed in LC-MS / MS are as follows: Sequence number 1 VNQIGSVTESIK SEQ ID NO:2 LAIMENC[CAM]NVLAR Although various methods can be used to detect these salmon-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 reverse phase solid phase column.

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

[0024] In the salmon detection method of the present invention, the type of test sample to be used is not particularly limited. For example, test samples include food ingredients and processed foods. Food ingredients include food ingredients that are produced separately in a food ingredient production factory that handles salmon and that do not intentionally contain salmon. Processed foods include confectionery, noodles, powdered soup, liquid soup, hot air dried or freeze-dried ingredients, or various cooked foods containing these processed foods. In addition, processed foods that are produced separately in a food production factory that handles salmon and that do not intentionally contain salmon are included. In addition, when producing processed foods that do not contain salmon after producing processed foods that contain salmon, it is essential to carefully clean the food production equipment with the removal of salmon residues in mind. From the viewpoint of confirming the effectiveness of this cleaning method and the presence or absence of salmon residues in the food production equipment, wiped samples of the production equipment can also be used as test samples.

[0025] Working Example The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. In addition, the present invention can be appropriately modified without departing from the gist of the present invention.

[0026] Example 1 Analysis of standard samples with known salmon protein concentrations In order to verify the quantitative capability of the salmon detection method by LC-MS / MS of the present invention, standard samples with known salmon protein concentrations were analyzed and a calibration curve was prepared.

[0027] Proteins were extracted from salmon 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.

[0028] Of the prepared standard samples, 40 μg of protein was taken into a 2.0 mL low-binding 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.

[0029] 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. Then, 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·alkylation).

[0030] 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 salmon standard sample.

[0031] 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 3 times.

[0032] The solution after surfactant removal was concentrated with a centrifugal evaporator, 0.1% formic acid was added, and purification was performed using a C18 reverse-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase.

[0033] The purified solution was dried by a centrifugal evaporator, dissolved in 0.1% formic acid containing 5% acetonitrile, and a dilution series with a concentration conversion of 1.25 - 20 μg / mL in the sample of total protein was prepared and analyzed by LC-MS / MS.

[0034] <LC-MS / MS device> 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℃ 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%) → Initialize <Mass spectrometry conditions> Ionization: Electrospray ionization Polarity: Positive Spray voltage: 5500 V

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

[0036] [Table 1]

[0037] FIG. 1 shows an example of a chromatogram obtained by analyzing a standard sample with a salmon total protein concentration of 1.25 μg / mL (peptide sequence: VNQIGSVTESIK (SEQ ID NO: 1), Q1: 637.9, Q3: 342.2).

[0038] A calibration curve under the same detection conditions as in Figure 1 is shown in Figure 2. A good calibration curve with R2: 0.986 was obtained in the range of 1.25 to 20 ppm, calculated as the concentration of salmon total protein in the sample.

[0039] Example 2 Salmon protein spike test for processed foods In order to examine the applicability of the salmon detection method by LC-MS / MS of the present invention to processed foods, a salmon-free white rice porridge was analyzed after adding a salmon protein standard sample to the rice porridge at 10 ppm.

[0040] 1 g of a white rice porridge sample not containing salmon was weighed into a 50 mL polypropylene centrifuge tube, and the salmon protein standard sample used in Example 1 was added to a total salmon protein concentration of 10 ppm.

[0041] 30 μL of ethylenediaminetetraacetic acid (EDTA) adjusted to 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 operations 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 white rice porridge not containing salmon is shown in Figure 3, and the chromatogram of a sample to which a salmon protein standard sample was added so that the product content was 10 ppm is shown in Figure 4 (peptide sequence: VNQIGSVTESIK (sequence number 1), Q1: 637.9, Q3: 342.2).

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

Claims

1. A method for detecting salmon, 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 salmon protein is 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 638 / 342, 638 / 820, 426 / 577, or 426 / 289 ii) SEQ ID NO:2, m / z values ​​of about 702 / 1107, 702 / 975, or 702 / 846 and determining qualitatively or quantitatively whether or not salmon 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 determining qualitatively or quantitatively whether or not salmon 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.

Images