Method for detecting mackerel using mass spectroscope

Abstract

To detect a mackerel 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 mackerel 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 4, using a mass spectroscope. The method thus determines whether or not a mackerel 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 mackerel using a mass spectrometer, which enables high sensitivity detection of even trace amounts of mackerel, which may cause food allergies, when it is contained in food ingredients, products, etc. [Background technology]

[0002] Mackerel (Scomber japonicus, etc.) is a fish of the Scomberidae family, and in Japan, it is designated as a "substance equivalent to a specific ingredient" that is recommended to be labeled as a substance that may 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 detection of mackerel, 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 objective of the present invention is to provide an analytical method using a mass spectrometer that can specifically and sensitively detect mackerel, 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 mackerel proteins to be detected and have intensively studied a method for detecting mackerel specifically and with high sensitivity. As a result, they have found amino acid sequences characteristic of mackerel, and found that by detecting these amino acid sequences, mackerel can be detected specifically and with high sensitivity, thus completing the present invention. That is, the present invention first relates to the following items.

[0011] Section 1. A method for detecting mackerel, 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 mackerel 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 4, a method is preferred in which the peptides are analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) and at least one or more precursor-product ion pair transitions having specific m / z values ​​associated with specific amino acid sequences are monitored. 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 625 / 663, 625 / 592, 625 / 479, 625 / 415, 625 / 516, 625 / 587 ii) SEQ ID NO:2, m / z values ​​of about 632 / 663, 632 / 835, 632 / 307, 632 / 187, 632 / 734, 422 / 479, 422 / 592, 422 / 297, 422 / 332, or 422 / 663 iii) SEQ ID NO:3, m / z values ​​of about 416 / 690, 416 / 381, 416 / 346, 416 / 284, or 416 / 546 iv) SEQ ID NO:4, m / z values ​​of about 681 / 227, about 681 / 398, about 681 / 483, about 681 / 964, 681 / 1136, 455 / 780, 455 / 292, 455 / 709, or 455 / 483 and determining whether or not mackerel 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. The method for detecting mackerel as described in claim 2, further comprising a step of qualitatively or quantitatively determining whether or not mackerel 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. Effect of the Invention

[0015] According to the present invention, it is possible to detect peptides derived from mackerel protein by LC-MS / MS analysis, and it is possible to carry out quality control inspections to determine whether the above-mentioned mackerel is mixed in or used in the test food raw materials or test food. It can also contribute to preventing allergies and investigating causative substances when allergic symptoms occur. [Brief description of the drawings]

[0016] [Figure 1] Peaks of peptides derived from mackerel proteins in a chromatogram obtained from a standard sample with known mackerel concentration [Diagram 2] A calibration curve was created by plotting the area of ​​peptides derived from mackerel protein in a chromatogram obtained from a standard sample with a known mackerel concentration against the known mackerel protein concentration in the standard sample. [Diagram 3] An exemplary chromatogram of a white rice porridge sample that does not contain mackerel [Figure 4] An exemplary chromatogram containing peptides derived from mackerel protein obtained from a white rice porridge sample spiked with mackerel protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention provides a method for detecting mackerel protein present in trace amounts in test samples such as food raw materials and processed foods. The method includes the steps of extracting proteins from the test samples, 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 the present invention 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: SEQ ID NO:1 DAEVTAALDGCK SEQ ID NO:2 DAEITAALDGCK SEQ ID NO:3 AAGSFDHK SEQ ID NO:4 LLGNPSADDMNSK Although various methods can be used to detect these mackerel-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 mackerel-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 mackerel proteins.

[0024] In the mackerel 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 mackerel and that do not intentionally contain mackerel. 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 mackerel and that do not intentionally contain mackerel are included. In addition, when producing processed foods that do not contain mackerel after producing processed foods that contain mackerel, it is essential to carefully clean the food production equipment with the removal of mackerel residues in mind. From the viewpoint of confirming the effectiveness of this cleaning method and the presence or absence of mackerel 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 mackerel standard samples with known protein concentrations In order to verify the quantitative capability of the mackerel detection method using LC-MS / MS of the present invention, standard samples with known mackerel protein concentrations were analyzed and a calibration curve was created.

[0027] Proteins were extracted from mackerel 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 sample, 40 μg of protein was placed in a 2.0 mL low-adsorption polypropylene tube, to which 1000 μg of egg-derived ovalbumin and 100 μg of bovine albumin were added, making the total solution volume 700 μL.

[0029] 70μL of 1M TEAB and 28μL of 1M DTT were added, and the mixture was left to stand at 75℃ for 15 minutes, then at room temperature for 30 minutes. Next, 56μL of 1M iodoacetamide solution prepared with distilled water was added, and the mixture was left to stand at room temperature for 45 minutes in the dark, and then 28μL of 1M DTT was added (reduction / alkylation).

[0030] After adding 10 μL of a trypsin solution derived from bovine pancreas adjusted to 20 mg / mL with 0.1% formic acid, the mixture was left to stand overnight at 37°C to perform enzymatic digestion of the mackerel standard sample.

[0031] The resulting enzyme digest was acidified by adding formic acid, and then ethyl acetate was added to remove the surfactant contained in the extraction solution by liquid-liquid partitioning. The 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 concentrate was purified using a C18 reversed-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase.

[0033] The refined solution was dried using a centrifugal evaporator, dissolved in 0.1% formic acid containing 5% acetonitrile, and a dilution series with a concentration conversion in the sample of 1.25 - 20 μg / mL in terms of total protein of mackerel was prepared and 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> 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

[0035] The sequences and MRM transitions of the peptide fragments derived from mackerel proteins targeted for detection are shown in Table 1.

[0036]

Table 1

[0037] The chromatogram when analyzing a standard sample with a total mackerel protein concentration of 1.25 μg / mL is illustrated in Figure 1 (peptide sequence: LLGNPSADDMNSK (SEQ ID NO: 4), Q1: 681.3, Q3: 482.7).

[0038] A calibration curve under the same detection conditions as in Figure 1 is shown in Figure 2. A good calibration curve with R2: 0.989 was obtained in the range of 1.25 to 20 ppm, calculated as the concentration of mackerel total protein in the sample.

[0039] Example 2 Mackerel protein spike test in processed foods In order to examine the applicability of the mackerel detection method of the present invention using LC-MS / MS to processed foods, a mackerel-free tomato sauce to which a mackerel protein standard sample had been added at 10 ppm was analyzed.

[0040] 1 g of a tomato sauce sample not containing mackerel was weighed into a 50 mL polypropylene centrifuge tube, and the mackerel protein standard sample used in Example 1 was added to give a total mackerel 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] Figure 3 shows a chromatogram of a white porridge sample that does not contain mackerel, and Figure 4 shows a chromatogram of a sample to which a mackerel protein standard sample was added to make the product content 10 ppm (peptide sequence: LLGNPSADDMNSK (sequence number 4), Q1: 681.3, Q3: 482.7).

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

Claims

1. A method for detecting mackerel, 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 4 using a mass spectrometer to qualitatively or quantitatively determine whether or not mackerel 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 625 / 663, 625 / 592, 625 / 479, 625 / 415, 625 / 516, 625 / 587 ii) SEQ ID NO:2, m / z values ​​of about 632 / 663, 632 / 835, 632 / 307, 632 / 187, 632 / 734, 422 / 479, 422 / 592, 422 / 297, 422 / 332, or 422 / 663 iii) SEQ ID NO:3, m / z values ​​of about 416 / 690, 416 / 381, 416 / 346, 416 / 284, or 416 / 546 iv) SEQ ID NO: 4, m / z values ​​of about 681 / 227, about 681 / 398, about 681 / 483, about 681 / 964, 681 / 1136, 455 / 780, 455 / 292, 455 / 709, or 455 / 483 and determining qualitatively or quantitatively whether or not mackerel 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. The method for detecting mackerel according to claim 2, further comprising a step of qualitatively or quantitatively determining whether or not mackerel 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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