Method of detecting banana using mass spectrometer

Abstract

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

Description

[Technical Field]

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

[0002] Bananas (Malus domestica) are a plant of the Musaceae family, and in Japan they 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 bananas, for example, the following prior art has been disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 4937306 [Non-patent literature]

[0008] [Non-Patent Document 1] Yumiko Sakai, Kimie Ishihata, Shigeru Nakano, Toshihiro Yamada, Takeo Yano, Kouji Uchida, Yoshiki Nakao, Atsuo Urisu, Reiko Adachi, Reiko Teshima, Hiroshi Akiyama, Specific Detection of Banana Residue in Processed Foods Using Polymerase Chain Reaction; J Agric Food Chem., 58(14): 8145-8151, 2010.

[0009] On the other hand, the patent and non-patent documents are directed to detecting genes, and other methods may also be 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 bananas, which may cause allergies, from food ingredients and products. [Means for solving the problem]

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

[0012] Section 1. A banana detection method 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 the peptide consisting of sequence number 1 using a mass spectrometer to qualitatively or quantitatively determine whether or not banana 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 585 / 457, 585 / 620, or 585 / 400 and determining whether banana 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:

[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 a step of qualitatively or quantitatively determining whether or not banana 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 banana protein-derived peptides by LC-MS / MS analysis, enabling quality control testing to determine whether the banana is present in or used in test food ingredients or test foods. It also contributes to preventing allergies and investigating the causative agent when allergic symptoms occur. [Brief explanation of the drawings]

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

[0018] The present invention provides a method for detecting trace amounts of banana protein 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 this method 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 GPIQISFNYNYGPAGR Although various methods can be used to detect these banana-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 banana-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 banana protein.

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

[0028] Proteins were extracted from bananas purchased from a store using MPEX PTS Reagents (60 mM SDC SLS / 50 mM TEAB) (manufactured by GL Sciences Inc.), and the total protein concentration was determined using a 2-D Quant Kit (manufactured by Cytiva) to prepare a standard sample.

[0029] Among the prepared standard samples, 40 μg of protein was collected into a 2.0 mL low-adsorption polypropylene tube, 100 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, 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 left standing at 37 °C overnight to perform enzymatic digestion of the banana 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 three times.

[0033] The solution after surfactant removal was concentrated using 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 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 total banana protein was prepared 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 Mobile phase A: 0.1% formic acid; Mobile phase 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 banana protein-derived peptide fragments used as detection targets are shown in Table 1. <SOP-

[0037]

Table 1

[0038] The chromatogram when analyzing a standard sample with a banana total protein concentration of 1.25 μg / mL is illustrated in Figure 1 (peptide sequence: GPIQISFNYNYGPAGR (SEQ ID NO: 1), Q1: 585.3, Q3: 400.2).

[0039] The calibration curve under the same detection conditions as in Figure 1 is illustrated in Figure 2. A good calibration curve with R2: 0.998 was obtained in the range of 1.25 - 10 ppm in terms of the converted value of the banana total protein concentration in the sample.

[0040] Example 2 Spiking of banana protein into processed foods To examine the applicability of the banana detection method by LC-MS / MS of the present invention to processed foods, a banana protein standard sample was added to banana-free white rice porridge to a concentration of 10 ppm, and then analyzed.

[0041] 1 g of a banana-free white rice porridge sample was weighed into a 50 mL polypropylene centrifuge tube, and the banana protein standard sample used in Example 1 was added to give a total banana 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 banana-free white rice porridge sample is shown in Figure 3, and the chromatogram of a sample to which a banana protein standard sample was added at 10 ppm is shown in Figure 4 (peptide sequence: GPIQISFNYNYGPAGR (sequence number 1), Q1: 585.3, Q3: 400.2).

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

Claims

1. A banana 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 the peptide consisting of sequence number 1 using a mass spectrometer to qualitatively or quantitatively determine whether or not banana 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 585 / 457, 585 / 620, or 585 / 400 and qualitatively or quantitatively determining whether or not banana 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 a step of qualitatively or quantitatively determining whether or not banana 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.

Images