Method for detecting walnuts using a mass spectrometer

Abstract

[Problem] To detect, with a high sensitivity and even in trace amounts, walnut (Juglans sp.) contained in foods and food products, which has the risk of causing allergic reactions. [Solution] Provided is a method for detecting walnut, 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 walnut protein is present in the sample.

Description

Technical Field

[0001] The present invention relates to a method for detecting walnuts using a mass spectrometer, which enables highly sensitive detection of even trace amounts of walnuts when they are contained in food ingredients, products, etc. that may cause food allergies.

Background Art

[0002] Walnuts (Juglans sp.) are plants of the genus Juglans in the walnut family, and in Japan, they are designated as "specified raw materials" for which labeling as substances that may cause food allergies is mandatory (Food Labeling Standards, March 30, 2015, Shokushokuhyohatsu No. 139).

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

[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 the ELISA method, Western blot method, and immunochromatography method, and methods for detecting characteristic DNA base sequences by the PCR method, etc.

[0005] In recent years, methods for detecting peptides derived from proteins characteristic of specific foods using a mass spectrometer have been reported. It is a technique capable of quantifying the protein 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 walnuts, for example, the following prior art of the applicant is disclosed.

Prior Art Documents

Patent Documents

[0007] [Patent Document 1] Patent No. 6570288

[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 walnuts, 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 allergen protein of walnuts to be detected and diligently researched a method that can detect walnuts specifically and with high sensitivity. As a result, they discovered amino acid sequences characteristic of walnuts and found that walnuts can be detected specifically and with high sensitivity by detecting these amino acid sequences, thus completing the present invention. In other words, the present invention relates first to the following items.

[0011] Section 1. A method for detecting walnuts, 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 walnut protein is present in the sample by 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.

[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 values ​​of approximately 460 / 390, 460 / 575, or 460 / 472 ii) Sequence ID 2, m / z values ​​of approximately 451 / 390, 451 / 175, or 451 / 196 iii) Sequence ID 3, m / z value of approximately 497 / 403 or 497 / 751 A method for detecting walnuts, comprising the step of determining whether or not walnut 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 walnuts according to claim 2, comprising the step of qualitatively or quantitatively determining whether or not walnut 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 walnut protein-derived peptides, and has the effect of enabling quality control inspections such as whether or not the above-mentioned walnuts are mixed in the test food raw materials or test foods, or whether they are used. In addition, 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 walnut protein-derived peptide in the chromatogram obtained from a standard sample with a known walnut concentration [Figure 2] Calibration curve created by plotting the area of walnut protein-derived peptide in the chromatogram obtained from a standard sample with a known walnut concentration and the known walnut protein concentration in the standard sample [Figure 3] Exemplary chromatogram of an instant noodle sample without walnuts [Figure 4] Exemplary chromatogram containing walnut protein-derived peptide obtained from an instant noodle sample spiked with walnut protein

Modes for Carrying Out the Invention

[0017] The present invention provides a method for detecting trace amounts of walnut 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 the extraction of protein 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 QSQQGQSR Sequence ID 2 ATVVVYVVEGTGR Sequence ID 3 ELSFNMPR Various methods can be used to detect these walnut-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, standard samples with known walnut-derived protein concentrations can be processed in the same way as the test samples, analyzed by LC-MS / MS, and a calibration curve can be created to perform quantitative analysis of walnut protein.

[0024] In the walnut 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 walnuts at food ingredient production plants that handle walnuts. 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 walnuts at food manufacturing plants that handle walnuts are also included. Furthermore, when manufacturing processed foods that do not contain walnuts after manufacturing processed foods that contain walnuts, thorough cleaning of the food manufacturing equipment with the removal of walnut residue in mind is essential. From the viewpoint of confirming the effectiveness of this cleaning method and the presence or absence of walnut 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 walnut samples with known protein concentrations To verify the quantitative accuracy of the LC-MS / MS walnut detection method of the present invention, standard samples with known walnut protein concentrations were analyzed, and a calibration curve was created.

[0027] Protein was extracted from walnuts 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 volume of the solution 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 walnut 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 three times.

[0032] The solution after surfactant removal was concentrated by a centrifugal evaporator, and after adding 0.1% formic acid, 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 1.25 - 10 μg / mL was prepared in terms of the sample concentration of the walnut total protein 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>[ \n 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 walnut protein-derived peptide fragments that were detected.

[0036] [Table 1]

[0037] Figure 1 shows an example of a chromatogram obtained by analyzing a standard walnut sample with a total protein concentration of 1.25 μg / mL (peptide sequence: ELSFNMPR (SEQ ID NO: 3), Q1: 497.2, Q3: 751.4).

[0038] Figure 2 shows an example of a calibration curve under the same detection conditions as in Figure 1. A good calibration curve with R2:0.997 was obtained in the range of 1.25 to 10 ppm, converted to a total walnut protein concentration in the sample.

[0039] Example 2 Walnut protein spike testing in processed foods To investigate the applicability of the LC-MS / MS walnut detection method of the present invention to processed foods, a sample of instant noodle pulverized material that did not contain walnuts was analyzed after adding a walnut protein standard sample to a concentration of 10 ppm.

[0040] One g of instant noodle pulverized material sample without walnuts was weighed into a 50 mL polypropylene centrifuge tube, and the walnut protein standard sample used in Example 1 was added to achieve a total walnut 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 an instant noodle sample without walnuts, and Figure 4 shows a chromatogram of a sample to which a standard walnut protein sample was added to a product content of 10 ppm (peptide sequence: ELSFNMPR (SEQ ID NO: 1), Q1: 497.2, Q3: 751.4).

[0046] The target peak was only detected when a walnut protein standard sample was added.

Claims

1. A method for detecting walnuts, 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 walnut protein is present in the sample by detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 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 values ​​of approximately 460 / 390, 460 / 575, or 460 / 472 ii) Sequence ID 2, m / z values ​​of approximately 451 / 390, 451 / 175, or 451 / 196 iii) Sequence ID 3, m / z value of approximately 497 / 403 or 497 / 751 A method for detecting walnuts, comprising the step of qualitatively or quantitatively determining whether walnut 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 walnuts according to claim 2, comprising the step of qualitatively or quantitatively determining whether or not walnut 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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