Method of detecting orange using mass spectrometer

Abstract

To enable high-sensitivity detection of orange that might cause allergies contained in food ingredients and products even if the amount is minute.SOLUTION: A method of detecting orange 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 orange protein in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] Oranges (Citrus sinensis) are a plant of the Rutaceae family, and in Japan are designated as "equivalent to specific ingredients" that are recommended to be labeled as substances 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 oranges, for example, the following prior art has been disclosed. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kyohei Kiyota, Kentaro Kawatsu, Junko Sakata, Masato Yoshimitsu, Kazuhiko Akutsu, Taro Satsuki-Murakami, Masami Ki, Keiji Kajimura, Tetsuo Yamano, Development of monoclonal antibody-based ELISA for the quantification of orange allergen Cit s 2 in fresh and processed oranges; Food Chem., 232(1): 43-48, 2017

[0008] On the other hand, this non-patent document uses the ELISA method, and other methods may also be used. 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 oranges, 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 have focused on the amino acid sequences in orange proteins to be detected and have conducted extensive research into methods for specifically and sensitively detecting oranges. As a result, they have found amino acid sequences characteristic of oranges, and have found that detecting these amino acid sequences enables specific and highly sensitive detection of oranges, thereby completing the present invention. That is, the present invention first relates to the following items.

[0011] Section 1. A method for detecting oranges, comprising 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 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 orange 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 416 / 573, 416 / 145, 831 / 711, or 831 / 667 ii) SEQ ID NO: 2, m / z values ​​of approximately 640 / 674, 640 / 802, 640 / 950 and determining whether orange 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 orange according to claim 2, further comprising a step of qualitatively or quantitatively determining whether orange 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 peptides derived from orange protein by LC-MS / MS analysis, thereby enabling quality control testing to determine whether or not the above-mentioned orange is mixed into or used in test food ingredients or test foods. It also contributes to the prevention of allergies and the investigation of causative substances when allergic symptoms occur. [Brief explanation of the drawings]

[0016] [Figure 1] Orange protein-derived peptide peaks in chromatograms obtained from standard samples with known orange concentrations [Figure 2] A calibration curve was prepared by plotting the area of ​​the peptides derived from orange protein in a chromatogram obtained from a standard sample with a known orange concentration against the known orange protein concentration in the standard sample. [Figure 3] Example chromatogram of a tomato sauce sample without orange [Figure 4] Illustrative chromatogram containing orange protein-derived peptides obtained from a tomato sauce sample spiked with orange protein. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a method for detecting trace amounts of orange protein present in test samples, such as raw food materials and processed foods. Specifically, the method includes the steps of extracting proteins from the test samples, obtaining enzymatic digests of the extracted proteins with protease, and analyzing the enzymatic digests by LC-MS / MS to obtain chromatograms 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 LQSFGYTEYFHNR Sequence number 2 DITFQNTAGPSK Although various methods can be used to detect these orange-derived peptides, a mass spectrometer is used in the present invention. 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 orange-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 orange protein.

[0024] The type of test sample to be used in the orange 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 intentionally produced without oranges at a food ingredient production factory that handles oranges. 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 intentionally produced without oranges at a food production factory that handles oranges. Furthermore, when producing processed foods that do not contain oranges after producing processed foods containing oranges, careful cleaning of the food production equipment is essential, with the removal of orange residue in mind. To verify the effectiveness of this cleaning method and the presence or absence of orange residues in the food production equipment, wiped samples from the production equipment can also be used as 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 standard samples with known orange protein concentrations To verify the quantitative capability of the LC-MS / MS method for detecting oranges of the present invention, standard samples with known concentrations of orange protein were analyzed and a calibration curve was prepared.

[0027] Proteins were extracted from oranges purchased from a store using MPEX PTS Reagents (60 mM SDC SLS / 50 mM TEAB) (GE Healthcare), 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 ovalbumin from eggs and 100 μg of albumin from cows 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 it was left standing at 37 °C overnight to perform enzymatic digestion of the orange 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 using a centrifugal evaporator, 0.1% formic acid was added, and purification was performed using a C18 reversed-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase.

[0033] The solution after purification was dried using a centrifugal evaporator, dissolved in 0.1% formic acid containing 5% acetonitrile, and a dilution series with a concentration conversion in the sample of 1.25 - 20 μg / mL of the total orange protein was prepared 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> 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 orange protein-derived peptide fragments targeted for detection are shown in Table 1.

[0036]

Table 1

[0037] The chromatogram when analyzing a standard sample with an orange total protein concentration of 1.25 μg / mL is illustrated in Figure 1 (peptide sequence: DITFQNTAGPSK (SEQ ID NO: 2), Q1: 639.8, Q3: 674.4).

[0038] The calibration curve under the same detection conditions as in Figure 1 is illustrated in Figure 2. A good calibration curve with R2: 0.991 was obtained in the range of 1.25 - 20 ppm in terms of the converted value of the orange total protein concentration in the sample.

[0039] Example 2 Orange protein spike test in processed foods To examine the applicability of the orange detection method by LC-MS / MS of the present invention to processed foods, an orange protein standard sample was added to an orange-free tomato sauce to a concentration of 10 ppm, and then analyzed.

[0040] 1 g of a tomato sauce sample not containing orange was weighed into a 50 mL polypropylene centrifuge tube, and the orange protein standard sample used in Example 1 was added to give a total orange 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] The chromatogram of a tomato sauce sample containing no orange is shown in Figure 3, and the chromatogram of a sample to which an orange protein standard sample was added to give a product content of 10 ppm is shown in Figure 4 (peptide sequence: DITFQNTAGPSK (sequence number 2), Q1: 639.8, Q3: 674.4).

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

Claims

1. A method for detecting oranges, 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 orange 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 416 / 573, 416 / 145, 831 / 711, or 831 / 667 ii) SEQ ID NO: 2, m / z values ​​of approximately 640 / 674, 640 / 802, 640 / 950 and qualitatively or quantitatively determining whether or not orange 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 orange 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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