Method of detecting salmon roe using mass spectrometer

Abstract

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

Description

[Technical Field]

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

[0002] Salmon roe is the roe of salmon or trout, and in Japan it is designated as a "substance equivalent to a specific ingredient" that is recommended to be labeled as a substance that can cause food allergies (Food Labeling Standards, Food and Nutrition Information 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 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 the detection of salmon roe, for example, the following prior art has been disclosed. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Yutaka Shimizu, Hiroshi Oda, Kohsuke Seiki, Hiroki Saeki, Development of an enzyme-linked immunosorbent assay system for detecting β'-component (Onk k 5), a major IgE-binding protein in salmon roe; Food Chem., 181: 310-317, 2015.

[0008] On the other hand, this non-patent document uses the ELISA method, and other methods may also be used. 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 roe, which may cause allergies, from food ingredients and products. [Means for solving the problem]

[0010] In order to achieve the above object, the present inventors focused on the amino acid sequence of the salmon roe protein to be detected and conducted extensive research into a method for specifically and highly sensitively detecting salmon roe. As a result, they discovered amino acid sequences characteristic of salmon roe, and found that detecting these amino acid sequences enables specific and highly sensitive detection of salmon roe, leading to the completion of the present invention. That is, the present invention first relates to the following items.

[0011] Section 1. A method for detecting salmon roe, comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein using 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, thereby qualitatively or quantitatively determining whether salmon roe 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 and 2, 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 using 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 491 / 795, 491 / 694, or 491 / 534 ii) SEQ ID NO: 2, m / z values ​​of about 465 / 782, 465 / 668, 465 / 473, 465 / 540, 465 / 253, 465 / 623, or 465 / 574 and determining whether or not a 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. The method for detecting salmon roe according to claim 2, further comprising a step of qualitatively or quantitatively determining whether salmon roe 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. [Effects of the Invention]

[0015] According to the present invention, LC-MS / MS analysis enables the detection of peptides derived from salmon roe protein, enabling quality control testing to determine whether salmon roe is present or used in test food ingredients or test foods. This can also contribute to the prevention of allergies and the investigation of causative substances when allergic symptoms occur. [Brief explanation of the drawings]

[0016] [Figure 1] Ikura protein-derived peptide peaks in a chromatogram obtained from a standard sample with known ikura concentrations [Figure 2] A calibration curve was created by plotting the area of ​​peptides derived from salmon roe protein in a chromatogram obtained from a standard sample with a known salmon roe concentration against the known salmon roe protein concentration in the standard sample. [Figure 3] An exemplary chromatogram of a rice porridge sample containing no salmon roe [Figure 4] An exemplary chromatogram containing salmon roe protein-derived peptides obtained from a rice porridge sample spiked with salmon roe protein. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a method for detecting trace amounts of salmon roe protein present in test samples, such as raw food materials and processed foods. Specifically, the method includes the steps of extracting protein from the test sample, obtaining an enzymatic digest of the extracted protein with a protease, and analyzing the enzymatic digest by LC-MS / MS to obtain a chromatogram of the target peptides. A preferred embodiment of the method according to this embodiment is 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 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.

[0021] The resulting enzymatic digests are preferably analyzed by LC-MS / MS after removing the surfactant and purifying using a reversed-phase solid-phase column.

[0022] The peptide sequences analyzed in LC-MS / MS are as follows: Sequence number 1 GQTC[CAM]GLC[CAM]GK SEQ ID NO: 2 DHASEQNHINVK Although various methods can be used to detect these salmon roe-derived peptides, a mass spectrometer is used in the present invention. 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 reversed-phase solid-phase column.

[0023] In addition, a standard sample with a known concentration of ikura-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, allowing for quantitative analysis of ikura protein.

[0024] The type of test sample to be tested in the salmon roe 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 that are intentionally produced without salmon roe at a food ingredient production factory that handles salmon roe. 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 that are intentionally produced without salmon roe at a food production factory that handles salmon roe. Furthermore, when producing processed foods that do not contain salmon roe after producing processed foods that contain salmon roe, careful cleaning of the food production equipment is essential, with the removal of salmon roe residue in mind. To verify the effectiveness of this cleaning method and the presence or absence of salmon roe residue in the food production equipment, wiped samples from the production equipment can also be used as test samples.

[0025] Example 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, the present invention can be modified appropriately without departing from the gist of the present invention.

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

[0027] Extract proteins using MPEX PTS Reagents (60 mM SDC SLS / 50 mM TEAB) (manufactured by GL Sciences Inc.) starting from the amount purchased from the store, determine the total protein concentration using the 2-D Quant Kit (manufactured by Cytiva), and use it as a standard sample.

[0028] Among the prepared standard samples, collect 40 μg of protein amount into a 2.0 mL low-adsorption polypropylene tube, add 1000 μg of ovalbumin from eggs and 100 μg of albumin from cows, and make the total solution volume 700 μL.

[0029] Add 70 μL of 1M TEAB and 28 μL of 1M DTT, let it stand at 75°C for 15 minutes, then let it stand at room temperature for 30 minutes. Next, add 56 μL of an Iodoacetamide solution prepared to 1M with distilled water, let it stand at room temperature in the dark for 45 minutes, and then add 28 μL of 1M DTT (reduction and alkylation).

[0030] Add 10 μL of a trypsin solution derived from bovine pancreas prepared to 20 mg / mL with 0.1% formic acid, then let it stand at 37°C overnight to perform enzymatic digestion of the standard sample of the amount.

[0031] After adding formic acid to the obtained enzymatic digest to make it acidic, add ethyl acetate and remove the surfactant contained in the extraction solution by liquid-liquid partitioning. The removal operation was repeated 3 times.

[0032] Concentrate the solution after surfactant removal using a centrifugal evaporator, add 0.1% formic acid, and then perform purification using a C18 reverse-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase.

[0033] Dry the solution after purification using a centrifugal evaporator, dissolve it in 0.1% formic acid containing 5% acetonitrile, and prepare a dilution series with a concentration conversion in the sample of total protein of 1.25 - 20 μg / mL and analyze it 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 C-18, 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 the proteins of interest are shown in Table 1.

[0036]

Table 1

[0037] The chromatogram when analyzing a standard sample with a total protein concentration of 1.25 μg / mL is illustrated in Figure 1 (peptide sequence: GQTC[CAM]GLC[CAM]GK (SEQ ID NO: 1), Q1: 490.7, Q3: 795.3).

[0038] The calibration curve under the same detection conditions as in Figure 1 is illustrated in Figure 2. A good calibration curve with R2: 0.977 was obtained in the range of total protein sample concentration conversion values of 1.25 - 20 ppm.

[0039] Example 2 Testing salmon roe protein spikes in processed foods To examine the applicability of the salmon roe detection method of the present invention using LC-MS / MS to processed foods, a salmon roe protein standard sample was added to white rice porridge containing no salmon roe at a concentration of 10 ppm and analyzed.

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

[0041] 30 μL of ethylenediaminetetraacetic acid (EDTA) prepared at 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 procedures 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 rice porridge sample that does not contain salmon roe, and Figure 4 shows a chromatogram of a sample to which a salmon roe protein standard sample was added to achieve a product content of 10 ppm (peptide sequence: GQTC[CAM]GLC[CAM]GK (sequence number 1), Q1: 490.7, Q3: 795.3).

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

Claims

1. A method for detecting salmon roe, 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, thereby qualitatively or quantitatively determining whether salmon roe 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 491 / 795, 491 / 694, or 491 / 534 ii) SEQ ID NO: 2, m / z values ​​of about 465 / 782, 465 / 668, 465 / 473, 465 / 540, 465 / 253, 465 / 623, or 465 / 574 and qualitatively or quantitatively determining whether or not a 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, the transition being selected from the group consisting of:

3. The method for detecting salmon roe according to claim 2, further comprising a step of qualitatively or quantitatively determining whether salmon roe 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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