Method of detecting yam using mass spectrometer

Abstract

To enable high-sensitivity detection of yam that might cause allergies contained in food ingredients and products even if the amount is minute.SOLUTION: A method of detecting yam 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 and 2 using a mass spectrometer so as to qualitatively or quantitatively determine the presence or absence of yam protein in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] Yamaimo (Dioscorea japonica, Dioscorea polystachya, etc.) is a plant of the Dioscoreaceae family, Dioscorea genus, and in Japan 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 the detection of yam, for example, the following prior art by the applicant has been disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 4937306

[0008] On the other hand, the patent document is directed to detecting genes, and other methods are possible. Summary of the Invention [Problem to be solved by the invention]

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

[0010] In order to achieve the above object, the present inventors focused on the amino acid sequences in yam proteins to be detected and conducted extensive research into methods for specifically and sensitively detecting yam. As a result, they discovered amino acid sequences characteristic of yam, and found that detecting these amino acid sequences enables specific and highly sensitive detection of yam, leading to the completion of the present invention. That is, the present invention first relates to the following items.

[0011] Section 1. A method for detecting yam, which comprises 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 and 2 using a mass spectrometer to qualitatively or quantitatively determine whether or not yam protein is present in the sample.

[0012] Next, as a method for detecting at least one or more peptides selected from the group consisting of SEQ ID NOs: 1 and 2, a method is preferred in which the peptides are analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) and at least one or more precursor-product ion pair transitions having specific m / z values ​​associated with specific amino acid sequences are monitored. That is, the present invention relates to the following item 2.

[0013] 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 580 / 626, 580 / 713, 580 / 841, 580 / 189, 580 / 416, 580 / 314, or 580 / 288 ii) SEQ ID NO: 2, m / z values ​​of about 581 / 975, 581 / 827, 581 / 664, 581 / 187, 581 / 488, 581 / 975, 581 / 334, 581 / 517, 1161 / 334, 1161 / 517, 1161 / 975, or 1161 / 388 and determining whether or not yam 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:

[0014] 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 method for detecting yam according to claim 2, further comprising a step of qualitatively or quantitatively determining whether or not yam 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]

[0015] The present invention enables the detection of yam protein-derived peptides by LC-MS / MS analysis, thereby enabling quality control testing to determine whether or not the yam is present in or used in test food ingredients or test foods. It can also contribute to the prevention of allergies and the investigation of causative agents when allergic symptoms occur. [Brief explanation of the drawings]

[0016] [Figure 1] Peaks of peptides derived from yam protein in a chromatogram obtained from a standard sample with known yam concentration [Figure 2] A calibration curve was created by plotting the area of ​​peptides derived from yam protein in a chromatogram obtained from a standard sample with a known yam concentration against the known yam protein concentration in the standard sample. [Figure 3] An exemplary chromatogram of a white rice porridge sample that does not contain yam [Figure 4] An exemplary chromatogram containing peptides derived from yam protein obtained from a rice porridge sample spiked with yam protein. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a method for detecting trace amounts of yam protein present in test samples such as food raw 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.

[0018] Proteins can be extracted from test samples using a buffer solution containing a surfactant or a commercially available protein analysis kit.

[0019] Preferably, the protein extract from the test sample is further reduced and alkylated to block thiol groups.

[0020] 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.

[0021] The resulting enzymatic digests are preferably analyzed by LC-MS / MS after removing the surfactant and purifying using a reversed-phase solid-phase column.

[0022] The peptide sequences analyzed in LC-MS / MS are as follows: Sequence number 1 GMEQSPIQLR Sequence number 2 SVFYFEQLK Although various methods can be used to detect these yam-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.

[0023] In addition, a standard sample with a known concentration of yam-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 yam protein.

[0024] The type of test sample to be used in the yam 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 yam in a food ingredient production factory that handles yam. 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 yam in a food production factory that handles yam. Furthermore, when producing processed foods that do not contain yam after producing processed foods that contain yam, careful cleaning of the food production equipment is essential, with the removal of yam residue in mind. To verify the effectiveness of this cleaning method and the presence or absence of yam residue in the food production equipment, wiped samples from the production equipment are also examples of test samples.

[0025] 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.

[0026] Example 1 Analysis of a standard sample with known yam protein concentration To verify the quantitative capability of the yam detection method by LC-MS / MS of the present invention, standard samples with known concentrations of yam protein were analyzed and a calibration curve was created.

[0027] Proteins were extracted from store-bought yams using MPEX PTS Reagents (60 mM SDC SLS / 50 mM TEAB) (GL Sciences), and the total protein concentration was determined using a 2-D Quant Kit (Cytiva) to serve as 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. Then, 56 μL of an Iodoacetamide solution prepared to 1M with distilled water was added, and after standing for 45 minutes at room temperature in the dark, 28 μL of 1M DTT was added (reduction·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 yam 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 reverse-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase. <00​​​​​​​​​​​​​​​​Column temperature: 40℃ 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

[0035] The sequences and MRM transitions of the peptide fragments derived from yam proteins that were the targets of detection are shown in Table 1.

[0036] [Table 1]

[0037] FIG. 1 shows an example of a chromatogram obtained by analyzing a standard sample containing 1.25 μg / mL of yam total protein (peptide sequence: GMEQSPIQLR (SEQ ID NO: 1), Q1: 579.8, Q3: 626.4).

[0038] Figure 2 shows an example of a calibration curve under the same detection conditions as Figure 1. A good calibration curve with R2: 0.999 was obtained in the range of 1.25 to 20 ppm, calculated as the concentration of total yam protein in the sample.

[0039] Example 2 Testing spiking yam protein into processed foods To examine the applicability of the yam detection method of the present invention using LC-MS / MS to processed foods, a yam protein standard sample was added to 10 ppm of white rice porridge containing no yam and analyzed.

[0040] 1 g of a yam-free white rice porridge sample was weighed into a 50 mL polypropylene centrifuge tube, and the yam protein standard sample used in Example 1 was added to give a total yam protein concentration of 10 ppm.

[0041] 30 μL of ethylenediaminetetraacetic acid (EDTA) prepared at 100 mg / mL with 1N sodium hydroxide solution was added.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Figure 3 shows a chromatogram of a white rice porridge sample that does not contain yam, and Figure 4 shows a chromatogram of a sample to which a yam protein standard sample was added to make the product content 10 ppm (peptide sequence: GMEQSPIQLR (sequence number 1), Q1: 579.8, Q3: 626.4).

[0046] The target peak was observed only when the yam protein standard sample was added.

Claims

1. A method for detecting yam, 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 and 2 using a mass spectrometer to qualitatively or quantitatively determine whether or not yam 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 580 / 626, 580 / 713, 580 / 841, 580 / 189, 580 / 416, 580 / 314, or 580 / 288 ii) SEQ ID NO: 2, m / z values ​​of about 581 / 975, 581 / 827, 581 / 664, 581 / 187, 581 / 488, 581 / 975, 581 / 334, 581 / 517, 1161 / 334, 1161 / 517, 1161 / 975, or 1161 / 388 and qualitatively or quantitatively determining whether or not yam 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. and a step of qualitatively or quantitatively determining whether or not yam 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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