A method for detecting abalone using a mass spectrometer

Abstract

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

Description

Technical Field

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

Background Art

[0002] Abalone (Haliotis discus discus, Haliotis discus hannai, etc.) is an animal of the genus Haliotis in the family Haliotidae, and in Japan, it is designated as "substances conforming to specific raw materials" recommended to be indicated as substances that may cause food allergies (Regarding food labeling standards, March 30, 2015, Food Labeling Notice No. 139).

[0003] Foods that may cause allergies can be accidentally mixed in trace amounts during production, distribution, and processing. Therefore, as providers of food raw materials or products, it is important to conduct quality control on whether they are mixed in.

[0004] As methods for inspecting the presence or absence of contamination with specific foods, there are methods for detecting characteristic proteins using antigen-antibody reactions such as the ELISA method, Western blot method, and immunochromatography method, and methods for detecting characteristic DNA base sequences by the PCR method, etc.

[0005] In recent years, a method for detecting peptides derived from proteins characteristic of specific foods using a mass spectrometer has been reported. It is a technique capable of quantifying the proteins in the target raw materials, and has the advantages of reducing false positive reactions that are likely to occur when using antigen-antibody reactions, and enabling simultaneous detection of multiple items.

[0006] As prior art related to the detection of abalone, for example, the following prior art is disclosed.

Prior Art Documents

[0007] [Patent Document 1] Patent No. 4439426 [Patent Document 2] Patent No. 5611510 [Non-patent literature]

[0008] [Non-Patent Document 1] Seung-Man Suh, Mi-Ju Kim, Hee-In Kim, Hyun-Joong Kim, and Hae-Yeong Kim, A multiplex PCR assay combined with capillary electrophoresis for the simultaneous detection of tropomyosin allergens from oyster, mussel, abalone, and clam mollusk species; Food Chem., 317(1): 126451, 2020

[0009] On the other hand, the literature in question focuses on detecting genes, and other methods are also possible. [Overview of the Initiative] [Problems that the invention aims to solve]

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

[0011] To achieve the above objectives, the present inventors focused on the amino acid sequence of the abalone protein to be detected and diligently researched a method that can detect abalone specifically and with high sensitivity. As a result, they discovered amino acid sequences characteristic of abalone and found that abalone can be detected specifically and with high sensitivity by detecting these amino acid sequences, thus completing the present invention. In other words, the present invention relates first to the following items.

[0012] Section 1. A method for detecting abalone, comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein using a proteolytic enzyme, and qualitatively or quantitatively determining whether or not abalone protein is present in a sample by analyzing the enzymatic digest and detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1-2 using a mass spectrometer.

[0013] Next, as a method for detecting at least one peptide selected from the group consisting of Sequence IDs 1 and 2 above, it is preferable to analyze 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 item 2 below.

[0014] Section 2. The process involves extracting proteins from a sample, obtaining an enzymatic digest of the extracted proteins using proteolytic enzymes, and analyzing the enzymatic digest by liquid chromatography-tandem mass spectrometry (LC-MS / MS), as follows: i) m / z values ​​of Array 1, approximately 608 / 243, 608 / 616, 608 / 729, 608 / 974, 608 / 845, 608 / 1103, 406 / 487, 406 / 243, 406 / 502, or 406 / 275 ii) m / z values ​​for sequence number 2, approximately 484 / 690, 484 / 375, 484 / 619, 484 / 246, 484 / 277, 967 / 375, 967 / 277, and 967 / 490. Determining whether a scallop protein is present in a sample by monitoring at least one or more precursor-product ion pair transitions having specific m / z values associated with specific amino acid sequences 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 item 3 below. Item 3. The scallop detection method according to claim 2, comprising a step of qualitatively or quantitatively determining whether a scallop 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 sequences.

Advantages of the Invention

[0016] According to the present invention, LC-MS / MS analysis enables the detection of scallop protein-derived peptides, and has the effect of enabling quality control inspections such as whether the above-mentioned scallops are mixed in or used in test food raw materials and test foods. It can also contribute to preventing allergies and investigating causative substances when allergic symptoms occur.

Brief Description of the Drawings

[0017] [Figure 1] Peak of scallop protein-derived peptide in the chromatogram obtained from a standard sample with a known scallop concentration [Figure 2] Calibration curve created by plotting the area of the scallop protein-derived peptide in the chromatogram obtained from a standard sample with a known scallop concentration and the known scallop protein concentration in the standard sample [Figure 3] Exemplary chromatogram of a white miso sample without scallops [Figure 4]Exemplary chromatogram containing abalone protein-derived peptides obtained from an abalone protein-spiked milk sample

Mode for Carrying Out the Invention

[0018] The present invention provides a method for detecting trace amounts of abalone protein mixed in test samples such as food raw materials and processed foods. That is, it is a method including a step of extracting protein from a test sample, a step of obtaining an enzyme digest of the extracted protein using a proteolytic enzyme, and a step of analyzing the enzyme digest by LC-MS / MS to obtain a chromatogram of the peptide to be analyzed. Hereinafter, preferred embodiments of the method according to the present embodiment will be described.

[0019] For protein extraction from a test sample, a buffer solution containing a surfactant, a commercially available protein analysis kit, or the like can be used.

[0020] The protein extract from the test sample is preferably further subjected to reduction-alkylation to block thiol groups.

[0021] The sample prepared as described above is treated with a proteolytic enzyme. Examples of the proteolytic enzyme used in the method of the present invention include trypsin, chymotrypsin, etc., and preferably trypsin. The treatment conditions may be appropriately selected according to the type of enzyme. By this enzyme treatment, the protein to be detected is decomposed and a plurality of peptides are generated.

[0022] The obtained enzyme digest is preferably analyzed by LC-MS / MS after removing the surfactant or purifying it using a reversed-phase solid-phase column.

[0023] The peptide sequences to be analyzed in LC-MS / MS are as follows. SEQ ID NO: 1 IEEDLNNLQK SEQ ID NO: 2 YIAEDAER Various methods can be used to detect these abalone-derived peptides, but in this invention, a mass spectrometer is used. Of these, methods utilizing liquid chromatography are particularly preferred. For example, methods using LC-MS or LC-MS / MS are available. In particular, it is preferable to remove surfactants from the obtained enzyme digest and purify it using a reversed-phase solid-phase column before analyzing it by LC-MS / MS.

[0024] Furthermore, standard samples with known abalone-derived protein concentrations can be processed in the same way as the test samples, analyzed by LC-MS / MS, and a calibration curve can be created to perform quantitative analysis of abalone protein.

[0025] In the abalone detection method of the present invention, the type of test sample is not particularly limited. For example, test samples include food ingredients and processed foods. Food ingredients include food ingredients that are intentionally produced separately at food ingredient production plants that handle abalone and do not contain abalone. Processed foods include confectionery, noodles, powdered soups, liquid soups, hot-air dried or freeze-dried ingredients, or various cooked foods containing these processed foods. Also, processed foods that are intentionally produced separately at food manufacturing plants that handle abalone and do not contain abalone are also included. Furthermore, when manufacturing processed foods that do not contain abalone after manufacturing processed foods that contain abalone, thorough cleaning of the food manufacturing equipment with the removal of abalone residue in mind is essential. From the viewpoint of confirming the effectiveness of this cleaning method and the presence or absence of abalone residue in the food manufacturing equipment, wipe samples from the manufacturing equipment can also be used as test samples.

[0026] Examples The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Furthermore, it is possible to modify the present invention as appropriate without departing from its spirit.

[0027] Example 1 Analysis of standard abalone samples with known protein concentrations To verify the quantitative accuracy of the LC-MS / MS abalone detection method of the present invention, a calibration curve was created by analyzing standard samples with known abalone protein concentrations.

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

[0029] From the prepared standard samples, 40 μg of protein was taken into a 2.0 mL low-adsorption polypropylene tube, and 1000 μg of egg-derived ovalbumin and 100 μg of bovine-derived albumin were added to make a total solution volume of 700 μL.

[0030] 70 μL of 1M TEAB and 28 μL of 1M DTT were added, and the mixture was allowed to stand at 75°C for 15 minutes, followed by standing at room temperature for 30 minutes. Then, 56 μL of iodoacetamide solution prepared to 1M with distilled water was added, and the mixture was allowed to stand at room temperature in the dark for 45 minutes, after which 28 μL of 1M DTT was added (reduction and alkylation).

[0031] After adding 10 μL of a bovine pancreas-derived trypsin solution prepared to 20 mg / mL with 0.1% formic acid, the mixture was left to stand overnight at 37°C, and then the abalone standard sample was enzymatically digested.

[0032] Formic acid was added to the obtained enzyme digest to make it acidic, and then ethyl acetate was added to remove the surfactant contained in the extract by liquid-liquid partitioning. The removal procedure was repeated three times.

[0033] The solution after surfactant removal was concentrated using a centrifugal evaporator, and after adding 0.1% formic acid, it was purified using a C18 reversed-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase.

[0034] The refined solution was dried using a centrifugal evaporator, dissolved in 0.1% formic acid containing 5% acetonitrile, and a dilution series with a concentration of 1.25 - 20 μg / mL in terms of the sample concentration of total abalone protein was prepared and analyzed by LC-MS / MS.

[0035] <LC-MS / MS instrument> LC section: ExionLC AD system (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 abalone protein-derived peptide fragments targeted for detection are shown in Table 1.

[0037]

Table 1

[0038] The chromatogram when analyzing a standard sample with a total abalone protein concentration of 1.25 μg / mL is illustrated in Figure 1 (peptide sequence: IEEDLNNLQK (SEQ ID NO: 1), Q1: 608.3, Q3: 243.1).

[0039] Figure 2 shows an example of a calibration curve under the same detection conditions as in Figure 1. A good calibration curve with R2:0.999 was obtained in the range of 1.25 to 20 ppm as the sample concentration of total abalone protein.

[0040] Example 2 Abalone protein spike testing in processed foods To investigate the applicability of the LC-MS / MS abalone detection method of the present invention to processed foods, abalone protein standard samples were added to plain white rice porridge without abalone to a concentration of 10 ppm and then analyzed.

[0041] One g of a white rice porridge sample without abalone was weighed into a 50 mL polypropylene centrifuge tube, and the abalone protein standard sample used in Example 1 was added to achieve a total abalone protein concentration of 10 ppm.

[0042] 30 μL of ethylenediaminetetraacetic acid (EDTA), prepared to a concentration of 100 mg / mL in a 1N sodium hydroxide solution, was added.

[0043] 9 mL of the extraction solution used in Example 1 was added, and the mixture was shaken overnight at 90-110 rpm to extract the protein.

[0044] The mixture was centrifuged at 4°C and 10,000xg for 30 minutes, and 700 μL of the supernatant was collected in a 2.0 mL low-adsorption polypropylene tube.

[0045] The subsequent steps were carried out in the same manner as in Example 1, and the final dissolved solution was analyzed by LC-MS / MS.

[0046] Figure 3 shows a chromatogram of a white rice porridge sample without abalone, and Figure 4 shows a chromatogram of a sample to which abalone protein standard sample was added to a product content of 10 ppm (peptide sequence: IEEDLNNLQK (SEQ ID NO: 1), Q1: 608.3, Q3: 243.1).

[0047] The target peak was only detected when a standard abalone protein sample was added.

Claims

1. A method for detecting abalone, comprising the steps of extracting protein from a sample, treating the extracted protein with a proteolytic enzyme to obtain an enzymatic digest, and analyzing the enzymatic digest to qualitatively or quantitatively determine whether or not abalone protein is present in the sample by detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 2 using a mass spectrometer.

2. The process involves extracting proteins from a sample, treating the extracted proteins with proteolytic enzymes to obtain an enzymatic digest, and analyzing the enzymatic digest by liquid chromatography-tandem mass spectrometry (LC-MS / MS), as follows: i) m / z values ​​for sequence number 1, approximately 608 / 243, 608 / 616, 608 / 729, 608 / 974, 608 / 845, 608 / 1103, 406 / 487, 406 / 243, 406 / 502, or 406 / 275 ii) m / z values ​​for sequence number 2, approximately 484 / 690, 484 / 375, 484 / 619, 484 / 246, 484 / 277, 967 / 375, 967 / 277, and 967 / 490 A method for detecting abalone, comprising the step of qualitatively or quantitatively determining whether or not abalone 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 the following.

3. A method for detecting abalone according to claim 2, comprising the step of qualitatively or quantitatively determining whether or not abalone protein is present in a sample by monitoring at least two precursor-product ion pair transitions having specific m / z values ​​associated with the specific amino acid sequence.

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