Method for detecting sesame using mass spectrometer

Abstract

[Problem] To detect sesame (Sesamum indicum), which have the risk of causing an allergic reaction, with high sensitivity when the sesame is contained in a food raw material, a product or the like, even if the content of the sesame is very small. [Solution] Provided is a method for detecting sesame, the method comprising a step for extracting a protein from a sample, a step for allowing the extracted protein to react with a protease to produce an enzymatically digested product, and analyzing the enzymatically digested product, wherein at least one peptide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:3 is detected using a mass spectrometer to qualitatively or quantitatively determine whether or not a sesame protein is present in the sample.

Description

Sesame detection method using a mass spectrometer

[0001] The present invention relates to a method for detecting sesame seeds using a mass spectrometer, which enables high-sensitivity detection of even minute amounts of sesame seeds that may cause food allergies when they are contained in food ingredients, products, etc.

[0002] Sesame (Sesamum indicum) is a plant of the Pedaliaceae family, and in Japan it is designated as a "substance equivalent to a specific allergen" that is recommended to be labeled as a substance that can cause food allergies (Food Labeling Standards, Notice No. 139 of the Food Safety and Health Administration, 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 sesame seeds, for example, the following prior art by the applicant has been disclosed.

[0007] Patent No. 5366887

[0008] On the other hand, the patent document is directed to detecting genes, and other methods are possible.

[0009] Therefore, an object of the present invention is to provide an analytical method using a mass spectrometer that can specifically and sensitively detect sesame, which may cause allergies, in food ingredients and products.

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

[0011] Item 1. A method for detecting sesame, comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein with a protease, analyzing the enzymatic digest, and detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 3 using a mass spectrometer to qualitatively or quantitatively determine whether or not sesame protein is present in the sample.

[0012] Next, a preferred method for detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 3 is to analyze the peptide 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 the following item 2.

[0013] Item 2. A method for detecting sesame, comprising the steps of extracting proteins from a sample, obtaining enzymatic digests of the extracted proteins with protease, and analyzing the enzymatic digests by liquid chromatography tandem mass spectrometry (LC-MS / MS) to determine whether or not sesame proteins are present in the 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: i) SEQ ID NO: 1, m / z values ​​of about 465 / 600, or 465 / 201; ii) SEQ ID NO: 2, m / z values ​​of about 541 / 605, 541 / 419, or 541 / 718; and iii) SEQ ID NO: 3, m / z values ​​of about 526 / 485, 526 / 272, or 526 / 556.

[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. Item 3. The method for detecting sesame according to claim 2, which comprises a step of qualitatively or quantitatively determining whether or not sesame 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.

[0015] According to the present invention, LC-MS / MS analysis can be used to detect peptides derived from sesame proteins, enabling quality control testing to determine whether or not the above-mentioned sesame is mixed into or used in test food ingredients or test foods. This method can also contribute to preventing allergies and investigating the causative substances when allergic symptoms occur.

[0016] Peaks of peptides derived from sesame proteins in a chromatogram obtained from a standard sample with a known sesame concentration. A calibration curve created by plotting the area of ​​peptides derived from sesame proteins in a chromatogram obtained from a standard sample with a known sesame concentration against the known sesame protein concentration in the standard sample. An exemplary chromatogram of a rice porridge sample containing no sesame. An exemplary chromatogram containing peptides derived from sesame proteins obtained from a rice porridge sample spiked with sesame proteins.

[0017] The present invention provides a method for detecting trace amounts of sesame protein present in test samples such as food raw materials and processed foods. Specifically, the method comprises the steps of extracting proteins from the test sample, obtaining enzymatic digests of the extracted proteins with proteases, and analyzing the enzymatic digests by LC-MS / MS to obtain chromatograms 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: SEQ ID NO: 1 ISGAQPSLR SEQ ID NO: 2 IQSEGGTTELWDER SEQ ID NO: 3 AFYLAGGVPR Although various methods can be used to detect these sesame-derived peptides, the present invention utilizes a mass spectrometer. Among these, methods using liquid chromatography are particularly preferred. Examples include methods using LC-MS and LC-MS / MS. In particular, it is preferable to analyze the obtained enzymatic digests by LC-MS / MS after removing the surfactant and purifying them using a reverse-phase solid-phase column.

[0023] In addition, a standard sample with a known concentration of sesame-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 to quantitatively analyze sesame protein.

[0024] The type of test sample to be used in the sesame 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 sesame and that are produced separately in a food ingredient production factory that handles sesame. 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 sesame and that are produced separately in a food production factory that handles sesame. Furthermore, when producing processed foods that do not contain sesame after producing processed foods that contain sesame, careful cleaning of the food production equipment is essential, with the removal of sesame residue in mind. To verify the effectiveness of this cleaning method and to confirm the presence or absence of sesame residue in the food production equipment, wiped samples from the production equipment are also examples of test samples.

[0025] 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 Sesame Protein Concentrations To verify the quantitative capability of the sesame detection method by LC-MS / MS of the present invention, standard samples with known sesame protein concentrations were analyzed and a calibration curve was created.

[0027] Proteins were extracted from sesame seeds 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, and 1000 μg of egg-derived ovalbumin and 100 μg of bovine albumin were added to bring the total solution volume to 700 μL.

[0029] 70 μL of 1M TEAB and 28 μL of 1M DTT were added, and the mixture was left to stand at 75°C for 15 minutes, followed by 30 minutes at room temperature. Next, 56 μL of 1M iodoacetamide solution prepared with distilled water was added, and the mixture was left to stand at room temperature in the dark for 45 minutes, after which 28 μL of 1M DTT was added (reduction and 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 standing overnight at 37°C to perform enzymatic digestion of the sesame standard sample.

[0031] Formic acid was added to the resulting enzyme digest to make it acidic, and then ethyl acetate was added to remove the surfactant contained in the extract solution by liquid-liquid partitioning. This 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 residue was purified using a C18 reversed-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase.

[0033] The purified solution was dried using a centrifugal evaporator and dissolved in 0.1% formic acid containing 5% acetonitrile. A dilution series of 1.25 to 20 μg / mL, equivalent to the total sesame protein concentration in the sample, 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> Analytical 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 analysis conditions> Ionization: Electrospray ionization Polarity: Positive Spray voltage: 5500 V

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

[0036]

[0037] The chromatogram obtained by analyzing a standard sample with a sesame total protein concentration of 1.25 μg / mL is shown in FIG. 1 (peptide sequence: ISGAQPSLR (SEQ ID NO: 1), Q1: 464.8, Q3: 201.1).

[0038] Figure 2 shows an example of a calibration curve under the same detection conditions as Figure 1. A good calibration curve with R2: 0.993 was obtained in the range of 1.25 to 20 ppm, calculated as the concentration of sesame total protein in the sample.

[0039] Example 2: Sesame protein spiking test in processed foods In order to examine the applicability of the sesame detection method by LC-MS / MS of the present invention to processed foods, a sesame-free white rice porridge was spiked with a sesame protein standard sample at 10 ppm and analyzed.

[0040] 1 g of sesame-free white rice porridge sample was weighed into a 50 mL polypropylene centrifuge tube, and the sesame protein standard sample used in Example 1 was added to give a total sesame 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] The chromatogram of a sesame-free white rice porridge sample is shown in Figure 3, and the chromatogram of a sample to which a sesame protein standard sample was added so that the product content was 10 ppm is shown in Figure 4 (peptide sequence: ISGAQPSLR (SEQ ID NO: 1), Q1: 464.8, Q3: 201.1).

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

Claims

1. A method for detecting sesame 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 3 using a mass spectrometer to qualitatively or quantitatively determine whether or not sesame protein is present in the sample.

2. A method for detecting sesame comprising the steps of extracting proteins from a sample, treating the extracted proteins with a protease to obtain an enzymatic digest, and analyzing the enzymatic digest by liquid chromatography tandem mass spectrometry (LC-MS / MS) to qualitatively or quantitatively determine whether or not sesame protein is present in the 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: i) m / z values ​​of about 465 / 600, or 465 / 201 for SEQ ID NO:1; ii) m / z values ​​of about 541 / 605, 541 / 419, or 541 / 718 for SEQ ID NO:2; and iii) m / z values ​​of about 526 / 485, 526 / 272, or 526 / 556 for SEQ ID NO:

3.

3. The method for detecting sesame according to claim 2, further comprising a step of qualitatively or quantitatively determining whether or not sesame 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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