How to measure the protein content of rice

Abstract

To provide a method that enables quantitative evaluation of protein contained in rice.SOLUTION: A method of measuring protein content in rice is provided, involving measuring content of peptide fragments of protein using liquid chromatography-mass spectrometry.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring the protein content in rice. [Background technology]

[0002] Conventionally, methods for quantitatively measuring the content of specific proteins contained in rice include polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), Western blotting analysis, etc. For example, Non-Patent Document 1 below discloses a method for quantitatively analyzing proteins contained in rice by analyzing prolamins, a type of storage protein, by SDS-PAGE. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Plant Cell Physiology (1987), vol. 28, p.1517-1527 Summary of the Invention [Problem to be solved by the invention]

[0004] Incidentally, the relationship between the protein content of rice and the taste of rice has been thought to be that the lower the overall protein content of rice, the better the taste and umami of the rice. However, according to the research of the present inventors, as described in the Reference Examples below, it has been found that the total protein content of rice does not necessarily correlate with the taste and umami of rice.

[0005] Therefore, in order to thoroughly investigate the relationship between the proteins contained in rice and the taste and umami of rice, it is necessary to investigate the contents of various proteins in rice in more detail and comprehensively, but the conventional methods mentioned above do not allow for an absolute quantitative evaluation.

[0006] The present invention has been made in view of the above circumstances, and aims to provide a method for enabling quantitative evaluation of proteins contained in rice. [Means for solving the problem]

[0007] The present invention provides a method for measuring the content of protein in rice, comprising measuring the content of peptide fragments of the protein using liquid chromatography-mass spectrometry.

[0008] The protein may be a storage protein.

[0009] The protein may be at least one selected from globulin, glutelin, and prolamine.

[0010] In the method for measuring a peptide fragment of the above protein, the protein may be a 19 kDa globulin and the peptide fragment may be a peptide fragment having the amino acid sequence of SEQ ID NO: 1, the protein may be glutelin A1 and the peptide fragment may be a peptide fragment having the amino acid sequence of SEQ ID NO: 2, the protein may be glutelin A2 and the peptide fragment may be a peptide fragment having the amino acid sequence of SEQ ID NO: 3, the protein may be glutelin A3 and the peptide fragment may be a peptide fragment having the amino acid sequence of SEQ ID NO: 4, the protein may be glutelin B and the peptide fragment may be a peptide fragment having the amino acid sequence of SEQ ID NO: 5, the protein may be glutelin D and the peptide fragment may be a peptide fragment having the amino acid sequence of SEQ ID NO: 6, or the protein may be prolamin 13a-1 and the peptide fragment may be a peptide fragment having the amino acid sequence of SEQ ID NO: 7. Effect of the Invention

[0011] According to the present invention, a method is provided that allows for the quantitative evaluation of protein contained in rice. [Brief description of the drawings]

[0012] [Figure 1] FIG. 2 is a graph showing the relationship between the amount of protein contained in rice and the sensory evaluation of the taste and umami of rice in a reference example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Preferred embodiments of the present invention will be described in detail below, however, the present invention is not limited to the following embodiments.

[0014] In this embodiment, the method for measuring the protein content in rice is to measure the content of peptide fragments of a protein using liquid chromatography mass spectrometry (LC / MS), i.e., includes a step of measuring the content of peptide fragments of a protein corresponding to a protein to be measured using liquid chromatography mass spectrometry (LC / MS).

[0015] The protein to be quantified in the protein contained in rice may be a protein called storage protein, which accounts for the majority of the protein contained in rice. Examples of storage proteins include alkali-soluble glutelin, saline-soluble globulin, and alcohol-soluble prolamin. Examples of globulin include 19 kDa globulin, 7S globulin, and 12S globulin. Examples of glutelin include glutelin A1, glutelin A2, glutelin A3, glutelin B (glutelin B1, glutelin B2, glutelin B4, glutelin B5), glutelin D, and glutelin C (indica). Examples of prolamin include prolamin 13a-1 and prolamin 13b-1.

[0016] The method for measuring the content of peptide fragments of a protein using liquid chromatography mass spectrometry is not particularly limited, but for example, the measurement can be performed by the following method.

[0017] First, rice is milled to obtain rice flour, from which proteins are extracted and purified, and the proteins are then fragmented by enzymatic treatment to obtain peptide fragments. The method for extracting and purifying proteins from rice flour is not particularly limited, and ordinary extraction and purification methods can be appropriately adopted. The enzymes used in the method for fragmenting proteins by enzymatic treatment can be appropriately selected depending on the target peptide fragments, and the target peptide fragments can be obtained by digestion with trypsin, for example. After the enzymatic treatment, surfactants may be removed and desalted as appropriate.

[0018] On the other hand, a sample peptide solution containing a peptide labeled with an isotope of known concentration and different in mass from the standard element is prepared as an internal standard. Such a labeled peptide may be synthesized as a peptide into which an isotope-labeled amino acid has been introduced by peptide synthesis using an amino acid previously labeled with an isotope, or may be synthesized by chemically synthesizing a peptide and then binding a labeling reagent having an isotope element to the peptide. When an isotope-labeled peptide is synthesized by binding a labeling reagent to a peptide, a labeling reagent having a corresponding standard element is also bound to the peptide fragment obtained by the above enzyme treatment. The element to be isotopically labeled may have a different mass from the standard element, and may be heavier or lighter than the standard element. Examples of such an isotope include, for example, the standard element. 12 It is an isotope of C 13 C. 14 C. 11 Standard elements such as C 14 It is an isotope of N. 15 Standard elements such as N 16 It is an isotope of O 17 However, it is preferable to use a carbon isotope, which is the most abundant element in peptide fragments, in order to maintain good measurement sensitivity. As a labeling reagent, for example, the TMT label kit manufactured by Thermo Scientific is commercially available.

[0019] Next, the solution containing the peptide fragments derived from rice protein obtained above is mixed with a standard peptide solution to prepare a measurement sample, and the content of the peptide fragments of the protein is measured by liquid chromatography-mass spectrometry.

[0020] The liquid chromatography mass spectrometry can be carried out, for example, using a liquid chromatograph mass spectrometer LC-MS9010 (manufactured by Shimadzu Corporation). The analysis conditions for the liquid chromatography mass spectrometry are, for example, as follows. Column: InertSustain AQ-C18 (φ2.1mm×150mm, GL Sciences Inc.) Column temperature: 40℃ Flow rate: 400μl / min Injection volume: 5μl Solvent: Mobile phase A: ultrapure water (for LCMS) + 0.1% formic acid, Mobile phase B: 100% acetonitrile (for LCMS) + 0.1% formic acid, 5% mobile phase B (0-2 min), 5-45% mobile phase B (2-13 min), 45-95% mobile phase B (13-16 min), 95% mobile phase B (16-18 min), 5% mobile phase B (18-20 min)

[0021] The peptide fragments contained in the measurement sample are separated by the liquid chromatography (LC) column and sequentially fed online into a mass spectrometer for analysis. The absolute amount of the peptide to be quantified in the measurement sample is measured from the peak area ratio between the peptide to be quantified and the stable isotope-labeled peptide as an internal standard in the obtained chromatogram and the calibration curve, and the content of the protein corresponding to each peptide fragment is calculated from the absolute amount of the peptide fragment obtained.

[0022] The type of peptide fragment obtained by fragmenting a protein to be quantified varies depending on the protein to be quantified, but usually, multiple (two or more) types of peptide fragments are obtained by enzymatically treating one type of protein. From the viewpoint of measuring the content of peptide fragments with higher sensitivity and achieving more quantitative protein quantification, it is preferable to select and use an optimal peptide fragment when quantifying a specific protein. Here, as a method for selecting an optimal peptide fragment, it is preferable to select one having a large peak area value and a sharp peak shape in the chromatogram obtained above, from the viewpoint of further increasing detection sensitivity. Preferred combinations of the protein to be quantified and the peptide fragments to be used are as follows.

[0023] When the protein to be quantified is a 19 kDa globulin, it is preferable to use a peptide fragment having the amino acid sequence of SEQ ID NO: 1, which is one of the fragments obtained by trypsin digestion of the 19 kDa globulin. When the protein to be quantified is glutelin A1, it is preferable to use a peptide fragment having the amino acid sequence of SEQ ID NO: 2, which is one of the fragments obtained by trypsin digestion of glutelin A1. When the protein to be quantified is glutelin A2, it is preferable to use a peptide fragment having the amino acid sequence of SEQ ID NO: 3, which is one of the fragments obtained by trypsin digestion of glutelin A2. When the protein to be quantified is glutelin A3, it is preferable to use a peptide fragment having the amino acid sequence of SEQ ID NO: 4, which is one of the fragments obtained by trypsin digestion of glutelin A3. When the protein to be quantified is glutelin B, it is preferable to use a peptide fragment having the amino acid sequence of SEQ ID NO: 4, which is one of the fragments obtained by trypsin digestion of glutelin B; when the protein to be quantified is glutelin D, it is preferable to use a peptide fragment having the amino acid sequence of SEQ ID NO: 6, which is one of the fragments obtained by trypsin digestion of glutelin D; and when the protein to be quantified is prolamin 13a-1, it is preferable to use a peptide fragment having the amino acid sequence of SEQ ID NO: 7, which is one of the fragments obtained by trypsin digestion of prolamin 13a-1.

[0024] Although the peptide fragments having the amino acid sequences of the above-mentioned SEQ ID NOs may have amino acid sequences other than the amino acid sequences of the respective SEQ ID NOs, it is preferable that the peptide fragments consist of the amino acid sequences of the respective SEQ ID NOs.

[0025] The origin of the rice used in this embodiment is not particularly limited, and any type of rice can be used. Examples of rice include Koshihikari, Sasanishiki, Hitomebore, Akitakomachi, and Tsubuzoroi. The origin of the rice is not particularly limited.

[0026] By measuring the protein content in rice using the method of this embodiment, the contents of various proteins in rice can be examined in more detail and comprehensively than with conventional methods. EXAMPLES

[0027] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples in any way.

[0028] (Reference example: Correlation between rice protein and taste / flavor) To investigate whether there is a correlation between the amount of protein contained in rice and its taste and umami, the protein value (%) of each brand of rice shown in Table 1 was measured using a taste analyzer (TM-3500, Shizuoka Seiki Co., Ltd.). In addition, each brand of rice shown in Table 1 was cooked and a sensory test was conducted by a randomly selected panel. The sensory test evaluated the taste and umami on a 7-point scale from 1 to 7. The higher the evaluation result, the better the taste and umami. Table 1 shows the protein value (%) and the average sensory evaluation value of each brand of rice, and Figure 1 shows the relationship between the protein value (%) of rice and the average sensory evaluation value of the taste and umami of rice.

[0029] [Table 1]

[0030] Regarding the relationship between the protein content of rice and the eating quality and umami of rice, it has been thought that the lower the overall protein content of rice, the higher the eating quality and umami of rice. However, as shown in Table 1 and Figure 1, it has been found that the amount of protein contained in rice does not necessarily correlate with the eating quality and umami of rice.

[0031] (Example 1: Measurement of 19 kDa globulin content in rice) <Protein extraction and purification> 50 mg of rice flour obtained by grinding rice (Koshihikari, Hitomebore, Sasanishiki, Akitakomachi and Tsubuzoroi) was weighed into a 2 ml tube, 1 ml of protein extraction buffer (buffer 950 μl (0.125 M Tris, 8 M urea, 4% SDS) + 2-mercaptoethanol 50 μl) was added, and the mixture was stirred at room temperature for more than 16 hours. After centrifugation at 16,000 × g for 15 minutes (4 ° C), the resulting supernatant (200 μl) was subjected to methanol chloroform precipitation to obtain a precipitate.

[0032] <Protein Fragmentation (Preparation of Peptide Fragments)> To the precipitate obtained above, 200 μl of protein dissolution buffer (50 mM TEAB (pH 8.5), 12 mM sodium deoxycholate monohydrate, 12 mM sodium N-lauroyl sarcosine) and 20 μl of 500 mM DTT solution were added, and the mixture was incubated at 37 ° C for 30 minutes, and then 20 μm of 500 mM IAA was added while shielding from light, and the mixture was incubated at room temperature for 30 minutes or more, and then 1 ml of 50 mM TEAB was added for dilution. Lys-C (Lysyl Endopeptidase (1 μg / μl)) was added at 2 μg per 100 μg of protein, and the mixture was incubated at 37 ° C for 4 hours, and then trypsin (1 μg / μl) was added at an equivalent amount of 1 μg per 50 μg of protein, and the mixture was incubated at 37 ° C for 18 hours or more to fragment the protein. After adding an equal volume of ethyl acetate to the solution containing the obtained peptide fragments, TFA was added to a final concentration of 0.5%, and the mixture was centrifuged (16000×g, 2 min), the organic layer was removed, and the mixture was dried under reduced pressure to obtain a pellet of the peptide fragments. The pellet was desalted using a spin column (Mono spin C18, GL Sciences Inc.), dried under reduced pressure, and redissolved in 100 μl of 0.2 mM TEMB to obtain a solution containing the peptide fragments.

[0033] <Labeling of peptide fragments> Labeling of peptide fragments was carried out using a TMT label kit (manufactured by Thermo Scientific). Specifically, 10 μl of TMT zero reagent was added to 100 μl of the solution containing the peptide fragments obtained above, and the mixture was reacted at room temperature for 1 hour. Then, 5% hydroxylamine (8 μl) was added to stop the reaction, thereby obtaining a solution containing TMT-labeled peptide fragments.

[0034] <Labeling of standard peptides> A solution (concentration 5.7 mg / ml) containing a peptide consisting of sequence number 1 was prepared, and a stable isotope was added to the solution. 13 Add TMT Lebel reagent containing C and react at room temperature for 1 hour, then add 5% hydroxylamine to stop the reaction and detect stable isotopes. 13Solution 1 containing a sample peptide labeled with TMT containing C was obtained.

[0035] <Measurement of 19 kDa globulin content by liquid chromatography-mass spectrometry> A 0.1% formic acid solution was added to each of the solution containing the peptide fragments obtained above and solution 1 containing the standard peptide, and 42.5 μl of the solution containing the peptide fragments and 7.5 μl of the solution containing the standard peptide were mixed and filtered through a filter (Ultrafree-MC-HV Centrifugal Filters Durapore PVDF 0.45 μm) to obtain a measurement sample.

[0036] The liquid chromatography mass spectrometry was carried out using a liquid chromatograph mass spectrometer LC-MS9010 (manufactured by Shimadzu Corporation) under the following analytical conditions. Column: InertSustain AQ-C18 (φ2.1mm×150mm, GL Sciences Inc.) Column temperature: 40℃ Flow rate: 400μl / min Injection volume: 5μl Solvent: Mobile phase A: ultrapure water (for LCMS) + 0.1% formic acid, Mobile phase B: 100% acetonitrile (for LCMS) + 0.1% formic acid, 5% mobile phase B (0-2 min), 5-45% mobile phase B (2-13 min), 45-95% mobile phase B (13-16 min), 95% mobile phase B (16-18 min), 5% mobile phase B (18-20 min)

[0037] The concentration of each peptide fragment contained in the sample was calculated from the peak area values ​​of the standard peptides with known concentrations obtained above and the peak area values ​​of the peptide fragments, and the concentration of the corresponding protein (19 kDa globulin) was calculated from the calculation results, which was 98.7 pmol.

[0038] (Example 2: Measurement of glutelin A1 content in rice) In labeling the sample peptide, a solution containing a peptide of SEQ ID NO:2 (concentration: 1.08 mg / ml) was prepared instead of a solution containing a peptide of SEQ ID NO:1. Solution 2 containing the sample peptide was prepared in the same manner as in Example 1 above, and the content of glutelin A1 in rice was measured. The calculated result was 59.9 pmol.

[0039] (Example 3: Measurement of glutelin A2 content in rice) In labeling the sample peptide, a solution containing a peptide of SEQ ID NO:3 (concentration: 5.0 mg / ml) was prepared instead of a solution containing a peptide of SEQ ID NO:1 in the same manner as in Example 1 above, except that a solution containing a sample peptide 3 was prepared and the content of glutelin A2 in rice was measured. The calculated result was 45.3 pmol.

[0040] (Example 4: Measurement of glutelin A3 content in rice) In labeling the sample peptide, a solution containing a peptide of SEQ ID NO: 4 (concentration: 5.0 mg / ml) was prepared instead of a solution containing a peptide of SEQ ID NO: 1, but in the same manner as in Example 1, a solution containing a sample peptide 4 was prepared and the content of glutelin A3 in rice was measured. The calculated result was 4.9 pmol.

[0041] (Example 5: Measurement of glutelin B content in rice) In labeling the sample peptide, a solution containing a peptide of SEQ ID NO:5 (concentration: 1 mg / ml) was prepared instead of a solution containing a peptide of SEQ ID NO:1. Solution 5 containing the sample peptide was prepared in the same manner as in Example 1 above, and the content of glutelin B in rice was measured. The calculated result was 12.4 pmol.

[0042] (Example 6: Measurement of glutelin D content in rice) In labeling the sample peptide, a solution containing a peptide of SEQ ID NO:6 (concentration: 5.0 mg / ml) was prepared instead of a solution containing a peptide of SEQ ID NO:1 in the same manner as in Example 1 above, except that a solution containing a sample peptide 6 was prepared and the content of glutelin D in rice was measured. The calculated result was 12.0 pmol.

[0043] (Example 7: Measurement of prolamin 13a-1 content in rice) In labeling the sample peptide, a solution containing a peptide of SEQ ID NO: 7 (concentration: 5.4 mg / ml) was prepared instead of a solution containing a peptide of SEQ ID NO: 1, but in the same manner as in Example 1, a solution containing a sample peptide 7 was prepared and the content of prolamin 13a-1 in rice was measured. The calculated result was 73.2 pmol.

[0044] In the following Test Examples 1 to 7, the measurement sensitivity of the content of peptide fragments was compared according to the type of peptide fragment obtained by fragmenting the protein to be quantified.

[0045] (Test Example 1: Comparison of measurement sensitivity by type of various peptide fragments of 19 kDa globulin) In labeling the sample peptides, solutions containing sample peptides of each peptide fragment were prepared in the same manner as in Example 1 above, except that, instead of preparing a solution containing a peptide consisting of SEQ ID NO: 1, solutions containing peptides corresponding to various peptide fragments of 19 kDa globulin were prepared as shown in Table 2 below. The obtained solutions containing the various sample peptides were measured using a liquid chromatograph mass spectrometer LC-MS9010 (manufactured by Shimadzu Corporation) under the same measurement conditions as those shown in Example 1 above, and the peak area values ​​and peak shapes were compared. The peptide fragments used and the peak area values ​​are shown in Table 2 below.

[0046] [Table 2]

[0047] In comparing the measurement sensitivity, it is preferable to select a peak having a large peak area and a sharp peak shape. Therefore, in comparing the measurement sensitivity of various types of peptide fragments of 19 kDa globulin, it is most preferable to use the peptide of SEQ ID NO: 1, taking into consideration the peak area and peak shape comprehensively.

[0048] (Test Example 2: Comparison of measurement sensitivity depending on the type of various peptide fragments of glutelin A1) In labeling the sample peptides, solutions containing sample peptides of each peptide fragment were prepared in the same manner as in Example 1 above, except that, instead of preparing a solution containing a peptide consisting of SEQ ID NO: 1, solutions containing peptides corresponding to various peptide fragments of glutelin A1 were prepared as shown in Table 3 below. The obtained solutions containing various sample peptides were measured using a liquid chromatograph mass spectrometer LC-MS9010 (Shimadzu Corporation) under the same measurement conditions as those shown in Example 1 above, and the peak area values ​​and peak shapes were compared. The peptide fragments used and the peak area values ​​are shown in Table 3 below.

[0049] [Table 3]

[0050] In comparing the measurement sensitivity, it is preferable to select a peak having a large peak area and a sharp peak shape. Therefore, in comparing the measurement sensitivity of various types of peptide fragments of glutelin A1, it is most preferable to use the peptide of SEQ ID NO: 2, taking into consideration the peak area and peak shape comprehensively.

[0051] (Test Example 3: Comparison of measurement sensitivity according to the type of various peptide fragments of glutelin A2) In labeling the sample peptides, solutions containing sample peptides of each peptide fragment were prepared in the same manner as in Example 1 above, except that, instead of preparing a solution containing a peptide consisting of SEQ ID NO: 1, solutions containing peptides corresponding to various peptide fragments of glutelin A2 were prepared as shown in Table 4 below. The obtained solutions containing the various sample peptides were measured using a liquid chromatograph mass spectrometer LC-MS9010 (manufactured by Shimadzu Corporation) under the same measurement conditions as those shown in Example 1 above, and the peak area values ​​and peak shapes were compared. The peptide fragments used and the peak area values ​​are shown in Table 4 below.

[0052] [Table 4]

[0053] In comparing the measurement sensitivity, it is preferable to select a peak having a large peak area and a sharp peak shape. Therefore, in comparing the measurement sensitivity of various types of peptide fragments of glutelin A2, it is most preferable to use the peptide of SEQ ID NO: 3, taking into consideration the peak area and peak shape comprehensively.

[0054] (Test Example 4: Comparison of measurement sensitivity depending on the type of various peptide fragments of glutelin A3) In labeling the sample peptides, solutions containing sample peptides of each peptide fragment were prepared in the same manner as in Example 1 above, except that, instead of preparing a solution containing a peptide consisting of SEQ ID NO: 1, solutions containing peptides corresponding to various peptide fragments of glutelin A3 as shown in Table 5 below were prepared. The obtained solutions containing the various sample peptides were measured using a liquid chromatograph mass spectrometer LC-MS9010 (Shimadzu Corporation) under the same measurement conditions as those shown in Example 1 above, and the peak area values ​​and peak shapes were compared. The peptide fragments used and the peak area values ​​are shown in Table 5 below.

[0055] [Table 5]

[0056] In comparing the measurement sensitivity, it is preferable to select a peak having a large peak area and a sharp peak shape. Therefore, in comparing the measurement sensitivity of various types of peptide fragments of glutelin A3, it is most preferable to use the peptide of SEQ ID NO: 4, taking into consideration the peak area and peak shape comprehensively.

[0057] (Test Example 5: Comparison of measurement sensitivity by type of various peptide fragments of glutelin B) In labeling the sample peptides, solutions containing sample peptides of each peptide fragment were prepared in the same manner as in Example 1 above, except that, instead of preparing a solution containing a peptide consisting of SEQ ID NO: 1, solutions containing peptides corresponding to various peptide fragments of glutelin B as shown in Table 6 below were prepared. The obtained solutions containing the various sample peptides were measured using a liquid chromatograph mass spectrometer LC-MS9010 (Shimadzu Corporation) under the same measurement conditions as those shown in Example 1 above, and the peak area values ​​and peak shapes were compared. The peptide fragments used and the peak area values ​​are shown in Table 6 below.

[0058] [Table 6]

[0059] In comparing the measurement sensitivity, it is preferable to select a peak having a large peak area and a sharp peak shape. Therefore, in comparing the measurement sensitivity of various types of peptide fragments of glutelin B, it is most preferable to use the peptide of SEQ ID NO: 5, taking into consideration the peak area and peak shape comprehensively.

[0060] (Test Example 6: Comparison of measurement sensitivity depending on the type of various peptide fragments of glutelin D) In labeling the sample peptides, solutions containing sample peptides of each peptide fragment were prepared in the same manner as in Example 1 above, except that, instead of preparing a solution containing a peptide consisting of SEQ ID NO:1, solutions containing peptides corresponding to various peptide fragments of glutelin D as shown in Table 7 below were prepared. The obtained solutions containing various sample peptides were measured using a liquid chromatograph mass spectrometer LC-MS9010 (manufactured by Shimadzu Corporation) under the same measurement conditions as those shown in Example 1 above, and the peak area values ​​and peak shapes were compared. The peptide fragments used and the peak area values ​​are shown in Table 7 below.

[0061] [Table 7]

[0062] In comparing the measurement sensitivity, it is preferable to select a peak having a large peak area and a sharp peak shape. Therefore, in comparing the measurement sensitivity of various peptide fragments of glutelin D, it is most preferable to use the peptide of SEQ ID NO: 6, taking into consideration the peak area and peak shape comprehensively.

[0063] (Test Example 7: Comparison of measurement sensitivity depending on the type of various peptide fragments of prolamin 13a-1) In labeling the sample peptides, solutions containing sample peptides of each peptide fragment were prepared in the same manner as in Example 1 above, except that, instead of preparing a solution containing a peptide consisting of SEQ ID NO: 1, solutions containing peptides corresponding to various peptide fragments of prolamin 13a-1 as shown in Table 8 below were prepared. The obtained solutions containing the various sample peptides were measured using a liquid chromatograph mass spectrometer LC-MS9010 (manufactured by Shimadzu Corporation) under the same measurement conditions as those shown in Example 1 above, and the peak area values ​​and peak shapes were compared. The peptide fragments used and the peak area values ​​are shown in Table 8 below.

[0064] [Table 8]

[0065] In comparing the measurement sensitivity, it is preferable to select a peak having a large peak area and a sharp peak shape. Therefore, in comparing the measurement sensitivity of various types of peptide fragments of prolamin 13a-1, it is most preferable to use the peptide of SEQ ID NO: 7, taking into consideration the peak area and peak shape comprehensively.

Claims

[Claim 1] A method for measuring the protein content of rice, comprising the steps of: The content of peptide fragments of the protein is measured using liquid chromatography-mass spectrometry; the protein is a 19 kDa globulin, and the peptide fragment has the amino acid sequence of SEQ ID NO: 1 or 11; the protein is glutelin A2, and the peptide fragment has the amino acid sequence of SEQ ID NO: 3 or 26; The protein is glutelin D, and the peptide fragment has the amino acid sequence of SEQ ID NO: 6 or 51; or The method, wherein the protein is prolamine 13a-1 and the peptide fragment is a peptide fragment having the amino acid sequence of SEQ ID NO:7.

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