Method of detecting peach using mass spectrometer

Abstract

To enable high-sensitivity detection of peach that might cause allergies contained in food ingredients and products even if the amount is minute.SOLUTION: A method of detecting peach 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 through 3 using a mass spectrometer so as to qualitatively or quantitatively determine the presence or absence of peach protein in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] Peach (Prunus persica) is a plant of the genus Prunus in the family Rosaceae, and in Japan it is designated as a "substance equivalent to a specific raw material" 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 peach detection, for example, the following prior art has been disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 5271556 [Non-patent literature]

[0008] [Non-Patent Document 1] Katsunari Ippoushi, Yoshimi Tanaka, Manabu Wakagi, Naoto Hashimoto, and Yuko Takano-Ishikawa, Assessment of Pru p 1 and Pru p 3 in peach fruit by liquid chromatography-tandem mass spectrometry; J. Food Compost. Anal., 80: 10-15, 2019. [Non-patent document 2] Wenhan Kang, Jiukai Zhang, Hong Li, Ning Yu, Rui Tang, Xiulan Sun, Liyang Wei, Jinlyu Sun, and Ying Chen, Quantification of major allergens in peach based on shotgun proteomics using liquid chromatography-tandem mass spectrometry; LWT Food sci. technol., 160: 113234, 2022.

[0009] Of the above prior art, Non-Patent Documents 1 and 2 are methods using mass spectrometry, but they do not disclose the detection sensitivity for the total protein content derived from raw materials, which is considered important in the labeling system in Japan. Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, an object of the present invention is to provide an analytical method using a mass spectrometer that can specifically and sensitively detect peaches, which may cause allergies, from food raw materials and products. [Means for solving the problem]

[0011] In order to achieve the above object, the present inventors have focused on the amino acid sequences of peach allergen proteins to be detected and have intensively studied methods for specifically and highly sensitively detecting peaches. As a result, they have found amino acid sequences characteristic of peaches, and have found that detecting these amino acid sequences enables specific and highly sensitive detection of peaches, thereby completing the present invention. That is, the present invention first relates to the following items.

[0012] Section 1. A peach detection method 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 to 3 using a mass spectrometer to qualitatively or quantitatively determine whether peach protein is present in the sample.

[0013] Next, as a method for detecting at least one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 3, a method is preferred in which the peptides are analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) to monitor at least one or more precursor-product ion pair transitions having specific m / z values ​​associated with specific amino acid sequences. That is, the present invention relates to the following item 2.

[0014] 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 401 / 587, 401 / 473, 401 / 214, or 401 / 246 ii) SEQ ID NO: 2, m / z values ​​of about 636 / 301, 636 / 477, 636 / 953, 636 / 660, 953 / 301, or 953 / 660 iii) SEQ ID NO: 3, m / z values ​​of about 651 / 756, 651 / 244, 651 / 219, 651 / 318, or 546 / 343 and determining whether peach 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:

[0015] 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 peach detection method described in claim 2, further comprising a step of qualitatively or quantitatively determining whether peach 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]

[0016] The present invention enables the detection of peach protein-derived peptides by LC-MS / MS analysis, thereby enabling quality control testing to determine whether the test food ingredients or test food contain or contain peaches, and also contributes to the prevention of allergies and the investigation of causative agents when allergic symptoms occur. [Brief explanation of the drawings]

[0017] [Figure 1]Peach protein-derived peptide peaks in chromatograms obtained from standard samples with known peach concentrations [Figure 2] A calibration curve was prepared by plotting the area of ​​peach protein-derived peptides in a chromatogram obtained from a standard sample with a known peach concentration against the known peach protein concentration in the standard sample. [Figure 3] Exemplary chromatogram of instant noodle sample without peaches [Figure 4] An exemplary chromatogram containing peptides derived from peach protein obtained from an instant noodle sample spiked with peach protein. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a method for detecting trace amounts of peach protein present in a test sample, such as a food ingredient or processed food. 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.

[0019] Proteins can be extracted from test samples using a buffer solution containing a surfactant or a commercially available protein analysis kit.

[0020] Preferably, the protein extract from the test sample is further reduced and alkylated to block thiol groups.

[0021] 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.

[0022] The resulting enzymatic digests are preferably analyzed by LC-MS / MS after removing the surfactant and purifying using a reversed-phase solid-phase column.

[0023] The peptide sequences analyzed in LC-MS / MS are as follows: SEQ ID NO: 1 NVNNLAR SEQ ID NO: 2 QLSASVPGVNPNNAAALPGK SEQ ID NO: 3 AFVLDADNLVPK Although various methods can be used to detect these peach-derived peptides, the present invention utilizes a mass spectrometer. 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.

[0024] In addition, a standard sample with a known concentration of peach-derived protein can be treated in the same manner as the test sample, analyzed by LC-MS / MS, and a calibration curve can be created to quantitatively analyze peach protein.

[0025] The type of test sample to be used in the peach 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 peaches at a food ingredient production factory that handles peaches. 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 peaches at a food production factory that handles peaches. Furthermore, when producing processed foods that do not contain peaches after producing processed foods that contain peaches, careful cleaning of the food production equipment is essential, with the removal of peach residue in mind. To verify the effectiveness of this cleaning method and the presence or absence of peach residue in the food production equipment, wipe samples from the production equipment are also examples of test samples.

[0026] 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.

[0027] Example 1 Analysis of standard samples with known peach protein concentrations To verify the quantitative capability of the peach detection method by LC-MS / MS of the present invention, standard samples with known concentrations of peach protein were analyzed and a calibration curve was prepared.

[0028] Protein was extracted from peaches 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 the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) to serve as a standard sample.

[0029] Among the prepared standard samples, 50 μg of protein was collected in 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.

[0030] 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. Then, 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).

[0031] 10 μL of a trypsin solution derived from bovine pancreas prepared to 20 mg / mL with 0.1% formic acid was added, and then it was left standing at 37 °C overnight to perform enzymatic digestion of the peach standard sample.

[0032] 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.

[0033] The solution after surfactant removal was concentrated with 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.

[0034] The purified solution was dried to dryness with a centrifugal evaporator, dissolved in 0.1% formic acid containing 5% acetonitrile, and a dilution series with a total protein concentration of 1.25 - 20 μg / mL was prepared for analysis by LC-MS / MS.

[0035] <LC-MS / MS device> LC section: ExionLC ADsystem (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 (5% B) → 16 min (40% B) → 18 min (95% B) → 23 min (95% B) → 23.1 min (5% B) → Initialization <Mass spectrometry conditions> Ionization: Electrospray ionization Polarity: Positive Spray voltage: 5500 V

[0036] The sequences and MRM transitions of the peach protein-derived peptide fragments that were the target of detection are shown in Table 1.

[0037] [Table 1]

[0038] FIG. 1 shows an example of a chromatogram obtained by analyzing a standard sample containing 1.25 μg / mL of peach total protein (peptide sequence: NVNNLAR (SEQ ID NO: 1), Q1: 400.7, Q3: 587.3).

[0039] Figure 2 shows an example of a calibration curve under the same detection conditions as Figure 1. A good calibration curve with R2: 0.987 was obtained in the peach total protein concentration range of 1.25 to 20 ppm.

[0040] Example 2 Testing the spike of peach protein into processed foods To examine the applicability of the peach detection method of the present invention using LC-MS / MS to processed foods, we analyzed instant noodle powder that did not contain peaches, to which a peach protein standard sample had been added at 10 ppm.

[0041] 1 g of a sample of instant noodle powder not containing peach was weighed into a 50 mL polypropylene centrifuge tube, and the peach protein standard sample used in Example 1 was added to give a total peach protein concentration of 10 ppm.

[0042] 30 μL of ethylenediaminetetraacetic acid (EDTA) prepared at 100 mg / mL with 1N sodium hydroxide solution was added.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Figure 3 shows a chromatogram of a sample of instant noodle powder that does not contain peaches, and Figure 4 shows a chromatogram of a sample to which a standard sample of peach protein was added to achieve a product content of 10 ppm (peptide sequence: NVNNLAR (sequence number 1), Q1: 400.7, Q3: 587.3).

[0047] The peak to be detected was observed only when the peach protein standard sample was added.

Claims

1. A peach detection method 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 peach 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 401 / 587, 401 / 473, 401 / 214, or 401 / 246 ii) SEQ ID NO: 2, m / z values ​​of about 636 / 301, 636 / 477, 636 / 953, 636 / 660, 953 / 301, or 953 / 660 iii) SEQ ID NO: 3, m / z values ​​of about 651 / 756, 651 / 244, 651 / 219, 651 / 318, or 546 / 343 and qualitatively or quantitatively determining whether or not peach 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:

3. The peach detection method of claim 2, further comprising a step of qualitatively or quantitatively determining whether peach 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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