Method for detecting cashew nuts using a mass spectrometer

Abstract

When cashew nuts, which may cause allergies, are contained in food ingredients or products, the system detects them with high sensitivity even in trace amounts. [Solution] A cashew nut detection method that includes 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 any of the peptides of SEQ ID NOs: 1 to 3 using a mass spectrometer to qualitatively or quantitatively determine whether or not cashew nut protein is present in the sample.

Description

Technical Field

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

Background Art

[0002] Cashew nuts (Anacardium occidentale) are plants of the genus Anacardium in the family Anacardiaceae and are designated as "substances equivalent to specific raw materials" for which labeling is recommended as substances that cause food allergies in Japan (Food Labeling Standards, March 30, 2015, Shokushihyohatsu No. 139). According to surveys conducted for the purpose of examining the validity and necessity of revisions of specific raw materials, etc., the top three causative foods of immediate-type food allergies have been eggs, milk, and wheat. However, in recent years, the number of cases caused by nuts has been increasing, and as a result, it is the third most common after excluding wheat (Report on the Research Project on Food Labeling Related to Food Allergies in 2021). Among nuts, cashew nuts are the second most common after walnuts and account for about 25% of nuts.

[0003] Foods that may cause allergies can be accidentally contaminated with extremely small 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] Examples of methods for inspecting the presence or absence of contamination with specific foods include 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.

[0005] In recent years, methods have been reported for detecting peptides derived from proteins characteristic of specific foods using mass spectrometry. This technique has the advantage of being able to quantify the protein of the target raw material, 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 relating to the detection of cashew nuts, for example, the following prior art by the applicants has been disclosed. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 6660504

[0008] On the other hand, the patent document in question focuses on detecting genes, and it is conceivable that other methods may also exist. [Disclosure of the Invention] [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 cashew nuts, 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 cashew nuts, which were the target of detection, and diligently researched a method that could specifically and sensitively detect cashew nuts. As a result, they discovered characteristic amino acid sequences of cashew nuts, and found that by detecting these amino acid sequences, cashew nuts could be detected specifically and sensitively, thus completing the present invention. In other words, the present invention relates first to the following items.

[0011] Section 1. A method for detecting cashew nut protein, comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein using a proteolytic enzyme, and analyzing the enzymatic digest to qualitatively or quantitatively determine whether cashew nut 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.

[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 452 / 304 or 452 / 391 ii) Sequence ID 2, m / z values ​​of approximately 445 / 346, 445 / 512, or 445 / 722 iii) Sequence ID 3, m / z values ​​of approximately 765 / 215, 765 / 362, or 765 / 490 A method for detecting cashew nuts, comprising the step of determining whether or not cashew nut 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 cashew nuts according to claim 2, comprising the step of qualitatively or quantitatively determining whether or not cashew nut 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 cashew nut protein-derived peptides, allowing for quality control inspections to determine whether or not cashew nuts are present in or used in the raw materials or food products of a test. Furthermore, it can contribute to the prevention of allergies and the investigation of causative substances when allergic symptoms occur. [Brief explanation of the drawing]

[0016] [Figure 1] Peak of cashew nut protein-derived peptide in chromatogram obtained from standard samples with known cashew nut concentrations. [Figure 2] A calibration curve was created by plotting the area of ​​cashew nut protein-derived peptides in chromatograms obtained from standard samples with known cashew nut concentrations against the known cashew nut protein concentrations in those standard samples. [Figure 3] Exemplary chromatogram of an instant noodle sample without cashews. [Figure 4] Exemplary chromatogram containing cashew nut protein-derived peptides obtained from instant noodle samples spiked with cashew nut protein. [Modes for carrying out the invention]

[0017] The present invention provides a method for detecting cashew nut protein mixed in trace amounts in a test sample such as a food raw material or processed food. 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 peptide to be analyzed. 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] The protein extract from the test sample is preferably further subjected to reduction-alkylation to block thiol groups.

[0020] The sample prepared as described above is treated with a proteolytic enzyme. Examples of the proteolytic enzyme used in the method of the present invention include trypsin, chymotrypsin, etc., and preferably trypsin. The treatment conditions may be appropriately selected according to the type of enzyme. By this enzyme treatment, the protein to be detected is decomposed to generate a plurality of peptides.

[0021] It is desirable that the obtained enzymatic digest be analyzed by LC-MS / MS after removing the surfactant or purifying it using a reversed-phase solid-phase column.

[0022] The peptide sequences to be analyzed in LC-MS / MS are as follows. SEQ ID NO: 1 GSESEEESEDEK SEQ ID NO: 2 GQVQVVDNFGNR SEQ ID NO: 3 DVFQQQQQHQSR As methods for detecting these cashew nut-derived peptides, various methods can be used, but in the present invention, a mass spectrometer is used. Among these, a method using liquid chromatography is particularly preferred. For example, methods using LC-MS or LC-MS / MS can be mentioned. 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 quantitatively analyze cashew nut protein by processing standard samples with known cashew nut protein concentrations in the same way as the test samples, analyzing them by LC-MS / MS, and creating a calibration curve.

[0024] In the cashew nut 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 cashew nuts at a food ingredient production plant that handles cashew nuts. 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 cashew nuts at a food manufacturing plant that handles cashew nuts are also included. Furthermore, when manufacturing processed foods that do not contain cashew nuts after manufacturing processed foods that contain cashew nuts, thorough cleaning of the food manufacturing equipment with the removal of cashew nut residue in mind is essential. From the viewpoint of confirming the effectiveness of this cleaning method and the presence or absence of cashew nut 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 cashew nut protein concentrations To verify the quantitative accuracy of the LC-MS / MS method for detecting cashew nuts according to the present invention, a calibration curve was created by analyzing standard samples with known cashew nut protein concentrations.

[0027] Protein was extracted from cashew nuts purchased from a store using MPEX PTS Reagents (60mM SDC SLS / 50 mM TEAB) (GL Sciences Inc.), and then tested with the Pierce BCA Protein Assay Kit. The total protein concentration was determined by Thermo Fisher Scientific and used as the standard sample.

[0028] From the prepared standard samples, 50 μg of protein was collected in a 2.0 mL low-adsorption polypropylene tube, and 1000 μg of egg-derived ovalbumin and 100 μg of bovine-derived albumin were added to make a total solution volume of 700 μL.

[0029] 70 μL of 1M TEAB and 28 μL of 1M DTT were added, and the mixture was allowed to stand at 75°C for 15 minutes, followed by standing at room temperature for 30 minutes. Then, 56 μL of iodoacetamide solution prepared to 1M with distilled water was added, and the mixture was allowed 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 bovine pancreas-derived trypsin solution prepared to 20 mg / mL with 0.1% formic acid, the mixture was left to stand overnight at 37°C, and then the cashew nut standard sample was enzymatically digested.

[0031] Formic acid was added to the obtained enzyme digest to make it acidic, and then ethyl acetate was added to remove the surfactant contained in the extract by liquid-liquid partitioning. The removal procedure was repeated three times.

[0032] The solution after surfactant removal was concentrated using a centrifugal evaporator, and after adding 0.1% formic acid, it 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 total cashew nut protein concentration of 1.25 - 10 μg / mL was prepared for analysis 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 cashew nut protein-derived peptide fragments targeted for detection are shown in Table 1.

[0036]

Table 1

[0037] The chromatogram when analyzing a standard sample with a total cashew nut protein concentration of 1.25 μg / mL is illustrated in Figure 1 (peptide sequence: GQVQVVDNFGNR (SEQ ID NO: 2), Q1: 444.9, Q3: 722.3).

[0038] Figure 2 shows an example of a calibration curve under the same detection conditions as in Figure 1. A good calibration curve with R2:0.994 was obtained in the range of 1.25 to 10 μg / mL for total cashew nut protein concentration.

[0039] Example 2 Cashew nut protein spike test on instant noodle pulp To investigate the applicability of the LC-MS / MS method for detecting cashew nuts according to the present invention to processed foods, a sample of instant noodle pulverized material that did not contain cashew nuts was analyzed after adding a standard cashew nut protein sample to a product content of 10 ppm, and the recovery rate was calculated.

[0040] One g of instant noodle pulverized material sample, which did not contain cashews, was weighed into a 50 mL polypropylene centrifuge tube, and the cashew nut protein standard sample used in Example 1 was added to achieve a total cashew nut 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 sample of instant noodle pulp without cashews, and Figure 4 shows a chromatogram of a sample to which a standard cashew nut protein sample was added to reach a product content of 10 ppm (peptide sequence: GQVQVVDNFGNR (SEQ ID NO: 2), Q1: 444.9, Q3: 722.3).

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

[0047] This application is based on Japanese application No. 2023-073864, filed on April 27, 2023, the contents of which are incorporated herein by reference.

Claims

1. A method for detecting cashew nut protein, comprising the steps of extracting protein from a sample, treating the extracted protein with a proteolytic enzyme to obtain an enzymatic digest, and qualitatively or quantitatively determining whether cashew nut protein is present in the sample by analyzing the enzymatic digest and detecting the peptide of Sequence ID No. 2 using a mass spectrometer.

2. A method for detecting cashew nut protein, 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 cashew nut protein is present in the sample by detecting the peptide of Sequence ID No. 1 using a mass spectrometer.

3. A method for detecting cashew nut protein, comprising the steps of extracting protein from a sample, treating the extracted protein with a proteolytic enzyme to obtain an enzymatic digest, and qualitatively or quantitatively determining whether cashew nut protein is present in the sample by analyzing the enzymatic digest and detecting the peptide of Sequence ID No. 3 using a mass spectrometer.

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