Method of detecting chicken using mass spectrometer

Abstract

To enable high-sensitivity detection of chicken that might cause allergies contained in food ingredients and products even if the amount is minute.SOLUTION: A method of detecting chicken 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 of SEQ ID NO: 1 using a mass spectrometer so as to qualitatively or quantitatively determine the presence or absence of chicken protein in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] Chickens (Gallus gallus domesticus) are animals of the genus Gallus in the family Phasianidae, and in Japan, their meat is designated as a "substance equivalent to a specific ingredient" 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 chicken meat, for example, the following prior art has been disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 3805692 [Non-patent literature]

[0008] [Non-Patent Document 1] Suyeon Sul, Mi-Ju Kim, Jung-Min Lee, Sung-Yeon Kim, and Hae-Yeong Kim, Development of a Rapid On-Site Method for the Detection of Chicken Meat in Processed Ground Meat Products by Using a Direct Ultrafast PCR System; J Food Prot., 83(6): 984-990, 2020

[0009] On the other hand, this document focuses on detecting genes, and other methods are possible. 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 chicken meat, which may cause allergies, in food raw materials and products. [Means for solving the problem]

[0011] In order to achieve the above object, the present inventors focused on the amino acid sequences of chicken proteins to be detected and conducted extensive research into methods for specifically and sensitively detecting chicken. As a result, they discovered amino acid sequences characteristic of chicken, and found that detecting these amino acid sequences enables specific and highly sensitive detection of chicken, leading to the completion of the present invention. That is, the present invention first relates to the following items.

[0012] Section 1. A method for detecting chicken meat, 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 the peptide consisting of sequence number 1 using a mass spectrometer to qualitatively or quantitatively determine whether or not chicken meat protein is present in the sample.

[0013] Next, a preferred method for detecting the peptide consisting of SEQ ID NO: 1 is to analyze it 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.

[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 442 / 505, 442 / 363, or 442 / 434 and determining whether chicken 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:

[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. and qualitatively or quantitatively determining whether or not chicken protein is present in the 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 peptides derived from chicken proteins by LC-MS / MS analysis, thereby enabling quality control testing to determine whether the test food ingredients or test food contain or contain chicken. This method can also contribute to the prevention of allergies and the investigation of causative substances when allergic symptoms occur. [Brief explanation of the drawings]

[0017] [Figure 1] Peaks of peptides derived from chicken protein in the chromatogram obtained from a standard sample with known chicken meat concentration [Figure 2] A calibration curve was prepared by plotting the area of ​​peptides derived from chicken protein in a chromatogram obtained from a standard sample with a known chicken protein concentration against the known chicken protein concentration in the standard sample. [Figure 3] An exemplary chromatogram of a white rice porridge sample without chicken [Figure 4] An exemplary chromatogram containing peptides derived from chicken protein obtained from a rice porridge sample spiked with chicken protein. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a method for detecting trace amounts of chicken protein present in test samples, such as raw food materials and processed foods. 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 ETFAAMGR Although various methods can be used to detect these peptides derived from chicken meat, 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 chicken-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 chicken protein.

[0025] The type of test sample to be used in the chicken meat 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 chicken meat at a food ingredient production factory that handles chicken meat. 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 chicken meat at a food production factory that handles chicken meat. Furthermore, when producing processed foods that do not contain chicken meat after producing processed foods that contain chicken meat, thorough cleaning of the food production equipment is essential, with the removal of chicken residue in mind. To verify the effectiveness of this cleaning method and to confirm the presence or absence of chicken 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 chicken meat standard samples with known protein concentrations In order to verify the quantitative capability of the chicken meat detection method by LC-MS / MS of the present invention, standard samples with known chicken meat protein concentrations were analyzed and a calibration curve was prepared.

[0028] Protein was extracted from chicken purchased from a store using MPEX PTS Reagents (60 mM SDC SLS / 50 mM TEAB) (GL Sciences Inc.), and the total protein concentration was determined using a 2-D Quant Kit (Cytiva) and used as a standard sample.

[0029] Among the prepared standard samples, 40 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 leaving it to stand at 75°C for 15 minutes, it was left to stand at room temperature for 30 minutes. Then, 56 μL of an iodoacetamide solution prepared to 1M with distilled water was added, and after leaving it to stand at room temperature in the dark for 45 minutes, 28 μL of 1M DTT was added (reduction·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 to stand at 37°C overnight to perform enzymatic digestion of the chicken 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 using a centrifugal evaporator, 0.1% formic acid was added, and purification was performed using a C18 reverse-phase solid-phase extraction centrifugal column and a silica gel-based anion-exchange solid phase.

[0034] The solution after purification was dried using a centrifugal evaporator, dissolved in 0.1% formic acid containing 5% acetonitrile, and a dilution series of 1.25 - 20 μg / mL was prepared as the sample concentration conversion of the total chicken protein and analyzed 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 (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

[0036] The sequences and MRM transitions of the peptide fragments derived from chicken proteins to be detected are shown in Table 1.

[0037]

Table 1

[0038] The chromatogram when analyzing a standard sample with a total chicken protein concentration of 1.25 μg / mL is illustrated in Figure 1 (peptide sequence: ETFAAMGR (SEQ ID NO: 1), Q1: 441.7, Q3: 505.3).

[0039] The calibration curve under the same detection conditions as in Figure 1 is illustrated in Figure 2. A good calibration curve with R2: 0.999 was obtained in the range of 1.25 - 20 ppm in terms of the converted value of the concentration in the sample of total chicken protein.

[0040] Example 2 Chicken protein spike test for processed foods To examine the applicability of the chicken meat detection method of the present invention using LC-MS / MS to processed foods, a chicken protein standard sample was added to a chicken-free rice porridge to a concentration of 10 ppm, and then analyzed.

[0041] 1 g of a white rice porridge sample not containing chicken meat was weighed into a 50 mL polypropylene centrifuge tube, and the chicken protein standard sample used in Example 1 was added to give a total chicken 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] The chromatogram of a white rice porridge sample containing no chicken is shown in Figure 3, and the chromatogram of a sample to which a chicken protein standard sample was added to make the product content 10 ppm is shown in Figure 4 (peptide sequence: ETFAAMGR (sequence number 1), Q1: 441.7, Q3: 505.3).

[0047] The target peak was observed only when the chicken protein standard sample was added.

Claims

1. A method for detecting chicken meat, 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 the peptide consisting of sequence number 1 using a mass spectrometer to qualitatively or quantitatively determine whether or not chicken meat 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 442 / 505, 442 / 363, or 442 / 434 and qualitatively or quantitatively determining whether or not chicken 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:

3. and qualitatively or quantitatively determining whether or not chicken protein is present in the 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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