Method for detecting sesame using a 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

Technical Field

[0001] The present invention relates to a method for detecting sesame using a mass spectrometer, which enables highly sensitive detection of even a trace amount of sesame when it is contained in food raw materials, products, etc. that may cause food allergies.

Background Art

[0002] Sesame (Sesamum indicum) is a plant of the genus Sesame in the Pedaliaceae family, and in Japan, it is designated as a "substance equivalent to specified raw materials" for which labeling is recommended as a substance that may cause food allergies (Food Labeling Standards, March 30, 2015, Food Labeling Notice No. 139).

[0003] Foods that may cause allergies can be accidentally contaminated with trace amounts during production, distribution, and processing. Therefore, as providers of food raw materials or products, it is important to conduct quality control to check whether they are contaminated.

[0004] As methods for inspecting the presence or absence of contamination with specific foods, there are methods for detecting characteristic proteins using antigen-antibody reactions such as ELISA, Western blotting, and immunochromatography, and methods for detecting characteristic DNA base sequences by PCR.

[0005] In recent years, methods for detecting peptides derived from proteins characteristic of specific foods using a mass spectrometer have been reported. This is a technique that can quantify the proteins of the target raw material, and has the advantages of reducing false positive reactions that are likely to occur when using antigen-antibody reactions, and enabling simultaneous detection of multiple items.

[0006] As prior art related to the detection of sesame, for example, the following prior art of the applicant is disclosed.

Prior Art Documents

Patent Documents

[0007] [Patent Document 1] Patent No. 5366887

[0008] On the other hand, the patent document in question targets genes for detection, but other methods are also possible. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Therefore, the objective of the present invention is to provide an analytical method using a mass spectrometer that can specifically and sensitively detect sesame seeds, which may cause allergies, from food ingredients and products. [Means for solving the problem]

[0010] To achieve the above objectives, the present inventors focused on the amino acid sequence of the sesame allergen protein to be detected and diligently researched a method for specifically and highly sensitively detecting sesame. As a result, they discovered characteristic amino acid sequences of sesame and found that sesame can be specifically and highly sensitively detected by detecting these amino acid sequences, thus completing the present invention. Specifically, the present invention relates first to the following items.

[0011] Section 1. A method for detecting sesame, comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein using a proteolytic enzyme, and qualitatively or quantitatively determining whether sesame protein is present in a sample by 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.

[0012] Next, as a method for detecting at least one peptide selected from the group consisting of Sequence IDs 1 to 3 above, it is preferable 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.

[0013] 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) Sequence ID 1, m / z value of approximately 465 / 600 or 465 / 201 ii) Sequence ID 2, m / z values ​​of approximately 541 / 605, 541 / 419, or 541 / 718 iii) Sequence ID 3, m / z values ​​of approximately 526 / 485, 526 / 272, or 526 / 556 A method for detecting sesame, comprising the step of determining whether or not sesame 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 the following.

[0014] 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 sesame according to claim 2, comprising the step of qualitatively or quantitatively determining whether or not sesame 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. [Effects of the Invention]

[0015] According to the present invention, LC-MS / MS analysis enables the detection of sesame protein-derived peptides, and has the effect of enabling quality control inspections such as whether the above-mentioned sesame is mixed in or used in the test food raw material or the 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] Peak of sesame protein-derived peptide in the chromatogram obtained from a standard sample with a known sesame concentration [Figure 2] Calibration curve created by plotting the area of the sesame protein-derived peptide in the chromatogram obtained from a standard sample with a known sesame concentration and the known sesame protein concentration in the standard sample [Figure 3] Exemplary chromatogram of a white sesame sample without sesame [Figure 4] Exemplary chromatogram containing a sesame protein-derived peptide obtained from a white sesame sample spiked with sesame protein

Modes for Carrying Out the Invention

[0017] The present invention provides a method for detecting trace amounts of sesame protein mixed in test samples such as food raw materials and processed foods. That is, it is a method including a step of extracting protein from the test sample, a step of obtaining an enzymatic digest of the extracted protein using a proteolytic enzyme, and a step of analyzing the enzymatic digest by LC-MS / MS to obtain a chromatogram of the target peptide. Hereinafter, preferred embodiments of the method according to the present embodiment will be described.

[0018] For protein extraction from the test sample, a buffer solution containing a surfactant, a commercially available protein analysis kit, or the like can be used.

[0019] It is preferable to further reduce and alkylate the protein extract from the test sample to block the thiol groups.

[0020] 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 and generates multiple peptides.

[0021] The obtained enzyme digest should preferably be purified by removing surfactants and using a reversed-phase solid-phase column before being analyzed by LC-MS / MS.

[0022] The peptide sequences analyzed by LC-MS / MS are as follows: Sequence ID 1 ISGAQPSLR Sequence ID 2 IQSEGGTTELWDER Sequence ID 3 AFYLAGGVPR Various methods can be used to detect these sesame-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.

[0023] Furthermore, it is possible to perform quantitative analysis of sesame protein by processing standard samples with known sesame protein concentrations in the same way as the test samples, analyzing them using LC-MS / MS, and creating a calibration curve.

[0024] In the sesame 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 sesame seeds at food ingredient production plants that handle sesame seeds. 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 sesame seeds at food manufacturing plants that handle sesame seeds are also included. Furthermore, when manufacturing processed foods that do not contain sesame seeds after manufacturing processed foods that contain sesame seeds, thorough cleaning of the food manufacturing equipment with the removal of sesame residue in mind is essential. From the viewpoint of confirming the effectiveness of this cleaning method and the presence or absence of sesame residue in the food manufacturing equipment, wipe samples from the manufacturing equipment can also be used as test samples.

[0025] Examples 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 present invention as appropriate without departing from its spirit.

[0026] Example 1 Analysis of standard samples with known sesame protein concentrations To verify the quantitative accuracy of the LC-MS / MS sesame detection method 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 (60mM SDC SLS / 50 mM TEAB) (GL Sciences), and the total protein concentration was determined using the 2-D Quant Kit (Cytiva) to create a standard sample.

[0028] Among the prepared standard samples, 40 μg of protein was taken into a 2.0 mL low-adsorption 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, 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 and 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 sesame 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 reversed-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 of 1.2~20 μg / mL in terms of the sample concentration of the sesame total protein was prepared and analyzed by LC-MS / MS.

[0034] <LC-MS / MS device> LC part: ExionLC AD system (SCIEX) MS / MS part: 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: 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%) → Initialize <Mass spectrometry conditions> Ionization: Electrospray ionization method Polarity: Positive Spray voltage: 5500 V

[0035] Table 1 shows the sequences and MRM transitions of the sesame protein-derived peptide fragments that were detected.

[0036] [Table 1]

[0037] Figure 1 shows an example of a chromatogram obtained by analyzing a standard sample with a total sesame protein concentration of 1.25 μg / mL (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 in Figure 1. A good calibration curve with R2:0.993 was obtained in the range of 1.25 to 20 ppm, converted to a total sesame protein concentration in the sample.

[0039] Example 2 Sesame protein spike testing in processed foods To investigate the applicability of the LC-MS / MS sesame detection method of the present invention to processed foods, a sesame protein standard sample was added to plain white rice porridge without sesame seeds to a concentration of 10 ppm and then analyzed.

[0040] One g of a 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 achieve a total sesame protein concentration of 10 ppm.

[0041] 30 μL of ethylenediaminetetraacetic acid (EDTA), prepared to a concentration of 100 mg / mL in a 1N sodium hydroxide solution, was added.

[0042] 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.

[0043] 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.

[0044] The subsequent steps were carried out in the same manner as in Example 1, and the final dissolved solution was analyzed by LC-MS / MS.

[0045] Figure 3 shows a chromatogram of a plain white rice porridge sample without sesame, and Figure 4 shows a chromatogram of a sample to which a standard sesame protein sample was added to a product content of 10 ppm (peptide sequence: ISGAQPSLR (SEQ ID NO: 1), Q1:464.8, Q3:201.1).

[0046] The target peak was only detected when a standard sesame protein 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 proteolytic enzyme to obtain an enzymatic digest; and analyzing the enzymatic digest to qualitatively or quantitatively determine whether sesame protein is present in the sample by detecting at least one peptide selected from the group consisting of SEQ ID NOs. 2 and 3 using a mass spectrometer.

2. 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) Sequence ID 1, m / z value of approximately 465 / 600 or 465 / 201 ii) m / z values ​​of approximately 541 / 605, 541 / 419, or 541 / 718 for sequence number 2. iii) Sequence ID 3, m / z values ​​of approximately 526 / 485, 526 / 272, or 526 / 556 A method for detecting sesame, comprising the step of qualitatively or quantitatively determining whether or not sesame 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 the following.

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

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