CN113514574A - Method for detecting allergen in high-oleic-acid peanuts by utilizing mass spectrometry - Google Patents
Method for detecting allergen in high-oleic-acid peanuts by utilizing mass spectrometry Download PDFInfo
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Abstract
The invention provides a method for detecting an allergen in high-oleic-acid peanuts by utilizing a mass spectrometry method, which comprises the following steps: carrying out mass spectrometry on the high oleic acid peanut extract by using a mass spectrometry parallel reaction monitoring method to obtain protein information; comparing and screening the obtained protein information with bioinformatics data in a UniProt database to obtain allergic protein related to peanut allergy; screening out specific peptide fragments in the sensitized protein through screening conditions; the screening conditions include: 1) no modification is carried out; 2) the number of the spectrogram matched with the amino acid sequence is more than 3 in the atlas database; 3) the sequence is present only in one or both proteins; 4) the number of amino acids is 7-20; 5) the peptide fragment does not contain cysteine and methionine. The method can qualitatively and quantitatively detect the peanut allergenic protein, and has high accuracy; and the protein and the peptide fragment can be confirmed, and the corresponding peptide fragment can be specifically screened out, so that a foundation is laid for detecting the peanut allergen by using an isotope dilution method subsequently.
Description
Technical Field
The invention belongs to the technical field of allergen detection, and particularly relates to a method for detecting allergen in high-oleic-acid peanuts by utilizing a mass spectrometry method.
Background
The high oleic acid peanuts are peanut varieties with the oleic acid content of more than 70 percent. The high-oleic-acid peanuts have the advantages that the content of linoleic acid is reduced while the content of oleic acid is increased, the ratio of the content of oleic acid to the content of linoleic acid is increased, the oxidation resistance of the peanuts is enhanced, and the storage stability is improved. At present, the related research on the sensitization of the high oleic acid peanuts is insufficient. There are two types of methods commonly used to detect peanut allergens at present: one is based on the detection method of allergen component genes, such as: polymerase chain reaction (PCR method), real-time fluorescent polymerase chain reaction (RT-PCR), and the like; the other is a method for detecting the sensitized protein, such as enzyme-linked immunosorbent assay (ELISA), Surface Plasmon Resonance (SPR), immunoblotting (WB), and the like.
The polymerase chain reaction method, namely the PCR amplification method, is a gene level allergen detection method based on the base complementary pairing principle in the DNA replication process, has the advantages of high sensitivity, high selectivity, strong specificity, no omission of allergen peanut-derived components, reliable safety guarantee and the like. A series of complex processing modes can be adopted in the actual processing and production process, the structure of the peanut allergenic protein can be damaged, the immunocompetence is reduced, and the test result can be deviated by using the traditional method. In this case, since the DNA of the peanut allergenic protein cannot be destroyed by ordinary processing treatment, the detection of DNA residues by the PCR method is more reliable than the direct detection of peanut allergenic protein. The PCR method has low requirement on the purity of the sample, and both the crude product of DNA and RNA can be directly used as amplification templates for detection. At present, the PCR method has some defects, and the PCR method can only discriminate whether peanut-derived components exist in the raw materials and cannot detect the peanut allergenic protein gene specifically; the PCR is used for detecting the peanut allergen, so that the phenomena of false negative and false positive are easy to occur; in addition, cross-contamination may occur during the experiment. These have limited the development of PCR methods for the detection of allergens.
Enzyme-linked immunosorbent assay (ELISA) has the advantages of rapid reaction, high sensitivity, strong pertinence and the like, and the ELISA is the most widely applied technology in the current immunological detection. However, the heating process can change the primary structure of the sensitized protein, so that the antibody cannot correctly identify the detected allergen, and a false negative phenomenon is generated. In addition, the food components are complex, protein denaturation is caused by processing, cross reaction also occurs in the detection mode of the ELISA antigen-antibody, the phenomena of high detection value and false positive occur, and the accuracy of the experimental result is influenced.
At present, the immunoblotting (WB) method is mature, and the immunoblotting method has many advantages in the aspect of detecting peanut allergenic protein, but because of the reasons of experimental operation, the phenomena of multiple bands, no bands, black spots on the background, uneven gel dyeing and the like easily occur, and the accuracy of the test is influenced, so the immunoblotting method is mostly used for qualitative analysis of the allergenic protein.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting allergens in high oleic acid peanuts by using a mass spectrometry, which is fast and accurate.
The invention provides a method for detecting an allergen in high-oleic-acid peanuts by utilizing a mass spectrometry method, which comprises the following steps:
carrying out mass spectrometry on the high oleic acid peanut extract by using a mass spectrometry parallel reaction monitoring method to obtain protein information;
comparing and screening the protein information with bioinformatics data in a UniProt database to obtain allergic protein related to peanut allergy;
screening out specific peptide fragments in the sensitized protein according to specific screening conditions to obtain specific peptide fragments in a specific allergen; the conditions for screening the sensitized protein containing the specific peptide fragment comprise the following steps: 1) without any modification (e.g., oxidation, deamination, and aminomethylation); 2) PSMs (peptides) with the length of amino acid sequence being more than 3, namely the number of the spectrogram matched with the amino acid sequence in the spectrogram library is more than 3; 3) the sequence is present only in one or both proteins; 4) the number of amino acids is 7-20; 5) the peptide fragment does not contain cysteine (C) and methionine (M).
In the present invention, the results of the mass spectrometry were retrieved using the Protome Discover software in the UniProt/Swiss-prot database.
In the present invention, the chromatographic column conditions for the mass spectrometry are: a C-18 chromatographic column; flow rate: 300 mu L/min; the sample introduction volume is 2 mL; solution A: 0.1% aqueous formic acid solution, solution B: 0.1% formic acid in acetonitrile, gradient elution;
mass spectrum conditions: electron source: ESI; the scanning mode is as follows: a PRM; adopting a positive ion mode, wherein the temperature of an ion transmission tube is 320 ℃; the primary full scanning range is 350-1550 m/z; the secondary scan AGC is set to 5.0e4, the ion implantation time IT is set to 100ms, and the collision energy is 32 eV.
In the invention, the high oleic peanut extract is prepared according to the following method:
peeling peanut, removing red skin, pulverizing, and defatting to obtain defatted peanut powder;
mixing the defatted peanut powder with a buffer solution, shearing at 8500-9500 rpm for 55-65 s, magnetically stirring at 4 ℃ for 4-4.2 h, centrifuging the obtained peanut protein homogenate, and collecting a supernatant;
boiling the supernatant, loading, performing electrophoresis for 50min by using a Bio-rad precast gel and a vertical electrophoresis tank at constant voltage of 150V, and decoloring after dyeing by using Coomassie brilliant blue to obtain a gel strip;
cutting the gel strip, chopping, decoloring with acetonitrile and ammonium bicarbonate, reducing disulfide bonds with dithiothreitol, performing enzymolysis with trypsin, treating at 35-40 ℃ for 10-15 h, then stopping the enzymolysis reaction, extracting the obtained peptide segment for multiple times with a dissolving solution containing 0.1% of trifluoroacetic acid and 50% of acetone, concentrating the extracted peptide segment, re-dissolving in a formic acid solution, and centrifuging to obtain the high-oleic-acid peanut extract.
In the invention, the molecular weight of the allergenic protein in the gel strip is 5 kDa-75 kDa.
In the present invention, the specific peptide fragment comprises one or more of Q9SQH7, B0YIU5, B6CG41, Q647G5, O82580 and Q45W 87. The specific peptide fragment preferably further comprises one or more of N1NG13, B0YIU5, B6CG41, Q647G5 and Q45W 87.
The invention provides a method for detecting an allergen in high-oleic-acid peanuts by utilizing a mass spectrometry method, which comprises the following steps: carrying out mass spectrometry on the high oleic acid peanut extract by using a mass spectrometry parallel reaction monitoring method to obtain protein information; comparing and screening the protein information with bioinformatics data in a UniProt database to obtain allergic protein related to peanut allergy; screening out specific peptide fragments in the sensitized protein according to conditions to obtain specific peptide fragments in the sensitized protein; the conditions for screening the specific peptide fragment in the sensitizing protein comprise: 1) without any modification (e.g., oxidation, deamination, and aminomethylation); 2) PSMs (peptides) with the length of amino acid sequence being more than 3, namely the number of the spectrogram matched with the amino acid sequence in the spectrogram library is more than 3; 3) the sequence is present only in one or both proteins; 4) the number of amino acids is 7-20; 5) the peptide fragment does not contain cysteine (C) and methionine (M). The method provided by the invention can be used for qualitatively and quantitatively detecting the peanut allergenic protein, and is high in accuracy. The method can solve the defects of low flux, cross interference and the like of an immunological method, can also solve the defect that a PCR method cannot directly detect the allergenic protein, can also confirm the protein and the peptide segment, and specifically screen out the corresponding peptide segment, thereby laying a foundation for identifying the peanut allergen by using an isotope dilution method subsequently.
Drawings
FIG. 1 is a diagram showing the distribution of each allergen component in electrophoresis in example 1 of the present invention.
Detailed Description
To further illustrate the present invention, a method for detecting allergens in high oleic peanuts by mass spectrometry according to the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
1. High oleic peanut allergen protein extraction
1.1 Experimental methods
a. Defatting peanut
Peeling peanut, removing red skin, and pulverizing into peanut paste. Mixing the peanut paste with acetone according to the mass-volume ratio of 1:10, carrying out magnetic stirring degreasing for 4 hours at 4 ℃, and then carrying out suction filtration to obtain a precipitate. The obtained precipitate is repeatedly degreased for 1 time, and is centrifugally separated to obtain degreased peanut powder.
b. Allergenic protein extraction
Mixing defatted peanut powder with Tris-HCl buffer solution containing 1M NaCl and pH8.00.05M at a mass-volume ratio of 1mg:10mL, shearing at 9000r/min for 1min, magnetically stirring at 4 deg.C for 4h, centrifuging peanut protein homogenate at 4 deg.C for 30min at 5000r/min, and collecting supernatant. And (4) roughly extracting the protein again for 1 time by using the precipitate, and collecting the supernatant obtained by secondary extraction.
c. Isolation of peanut allergen proteins
SDS-PAGE: dissolving the high oleic peanut extract with 1mL 2X SDS loading buffer, centrifuging, transferring the supernatant to a clean EP tube, boiling for 5min, loading 2 μ L, performing electrophoresis with Bio-rad precast gel and vertical electrophoresis tank at constant voltage 150V for 50min, and decolorizing after Coomassie brilliant blue staining.
1.2 analysis and discussion of results
The distribution of each allergen component in an electrophoretogram of the supernatant and the precipitate obtained after two times of extraction is shown in figure 1, and figure 1 is an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretogram of the supernatant and the precipitate of peanut protein obtained after two times of crude extraction, wherein 1. the sample adding amount is 1 mu L of the supernatant obtained by the first time of crude extraction; 2. adding 2 mu L of supernatant obtained by the first crude extraction; 3. adding 2 mu L of supernatant obtained by the second coarse extraction; 4. adding 4 mu L of supernatant obtained by the second coarse extraction; 5. adding 2 mu L of a first crude extraction precipitate; 6. adding 4 mu L of a first crude extraction precipitate; 7. adding 5 mu L of sample for the second crude extraction of the sediment; 8. the sample was added in an amount of 10. mu.L for the second crude extraction of the precipitate. Wherein the major allergenic proteins are labeled by molecular weight: ara h1(63.5kDa), Ara h2(17kDa), Ara h6(15 kDa). As can be seen from FIG. 1, Ara h1, Ara h2 and Ara h6 were extracted from both crude extractions. The content of the precipitate is less, which proves that the extraction method is effective.
2. Peanut allergen protein identification
2.1 Experimental methods
a. Gel trypsinization
The gel obtained after SDS-PAGE, the corresponding protein band, was cut off and further cut to 1mm3Small pieces, transferred in EP tubes, destained with acetonitrile and ammonium bicarbonate, disulfide threitol (DTT) reduced disulfide bonds, and eggsCarrying out enzymolysis with white enzyme, carrying out overnight treatment at 37 ℃, then terminating the enzymolysis reaction with 10% trifluoroacetic acid, extracting the obtained peptide fragment twice with a solution containing 0.1% trifluoroacetic acid and 50% acetone, concentrating the extracted peptide fragment with a vacuum centrifugal concentrator, redissolving with 0.1% formic acid ultrapure water, and carrying out high-speed centrifugation and uniform mixing for mass spectrometry.
b. Mass spectrometric detection
The allergens in high oleic peanut extracts were characterized using Thermo Orbitrap Fusion mass spectrometry Parallel Reaction Monitoring (PRM). Chromatographic column conditions: c-18 column (75. mu. m.times.2 cm,5 μm, Varian, Lexington, MA). Flow rate: 300 mu L/min; the sample introduction volume is 2 mL; solution A: 0.1% aqueous formic acid solution, solution B: 0.1% formic acid in acetonitrile, gradient elution.
Mass spectrum conditions: electron source: ESI; the scanning mode is as follows: a PRM; adopting a positive ion mode, wherein the temperature of an ion transmission tube is 320 ℃; the primary full scan range position is 350-1550 m/z; the AGC of the secondary scanning (dd-MS2) is set to 5.0e4, the ion injection time IT is set to 100MS, the collision energy is 32eV, and all mass spectrum results are searched in a Uniprot/Swiss-prot database by using the software of a Proteome Discover (Version 1.4) to obtain the most appropriate identification result.
2.2 Mass Spectrometry results and analysis
And (4) carrying out mass spectrometry after the electrophoresis strip is hydrolyzed by enzyme to obtain a result. 1159 proteins are separated by using mass spectrometry, wherein 752 proteins score more than or equal to 5 (response intensity in mass spectrometry is more than or equal to 5), and reliability is high. The obtained protein is compared and screened with bioinformatics data in a UniProt database, and 16 proteins or protein subunits related to peanut allergy are screened from 752 proteins. Of these, 2 were from Ara h1, 2 were from Ara h2, 3 were from Ara h3, 3 were from Ara h4, 2 were from Ara h6, and the remaining four were from Ara h8(17kD), Ara h10(16kD), Ara h11(14kD), and Ara h14(17.5kD), respectively.
Aiming at the relevant proteins to be screened, specific peptide fragments are screened according to the following conditions: 1) without any modification, such as oxidation, deamidation, etc.; 2) PSMs are more than 3, namely the number of the spectrogram matched with the amino acid sequence in the spectrogram library is more than 3; 3) no more than 2 corresponding proteins, i.e., the sequence is present only in one or both proteins; 4) the number of amino acids is 7-20; 5) the peptide fragment does not contain cysteine (C) and methionine (M).
Ji flower 5(JH5) is common peanut olein, and it can be seen from Table 1 that 76 sequences of 21 protein subunits of 14 peanut allergenic proteins meet the conditions after screening. The comparison literature finds that Q9SQH7, B0YIU5, B6CG41 and Q647G5 have been reported in the prior literature. Comparison with the previous literature shows that NNPFYFPSR, IVQIEAKPNTLVLPK, GSEEEGDITNPINLR, EGEQEWGTPGSHVR, RPSHQQPR, EEEEDEDEEEEGSNR, SSENNEGVIVK, EGEPDLSNNFGK, NNPFYFPSR, EHVEELTK in N1NG13 and FFVPPFQQSPR in Q9SQH7 (marked in bold in Table 1) have been reported, and therefore the feasibility of the screening method can be verified. In addition, the method also screens 3 unreported specific peptide fragments (DGEPDLSNNFGR, KGSEEEGDITNPINLR, NNPFYFPSRR) in the sensitizing protein Ara h1(N1NG13), and the protein subunits such as the sensitizing protein Ara h 8(B0YIU5), Ara h 9(B6CG41), Ara h10 (Q647G5) and the like.
TABLE 1 Ji flower 5 sensitized protein subunit and its specific peptide fragment
Ji flower 13(JH13) is high-oleic peanut, and as can be seen from Table 2, 64 sequences of 16 protein subunits in 11 peanut allergenic proteins meet the conditions after screening. The comparative literature shows that 18 peptide fragments of subunits Q9SQH7, B0YIU5, Q647G5 and the like are reported in the prior literature, such as peptide fragment FFVPPFQQSPR and the like in Q9SQH7 (bold marks are added in Table 2), so that the feasibility of the screening method can be verified. In addition, 5 unreported specific peptide fragments (GRREEEEDEDEEEEGSNR, VSKEHVEELTK, KGSEEEGDITNPINLR, NNPFYFPSRR, EHVEELTK) in the allergen protein Ara h1(N1NG13) are screened by the method, and unreported specific peptide fragments are also screened in other allergen proteins and subunits.
TABLE 2 JIHUA 13 sensitized protein subunit and its specific peptide fragment
Ji Hua 16(JH16) is high-oleic peanut, and it can be seen from Table 3 that 68 sequences of 18 protein subunits in 8 peanut allergenic proteins meet the conditions after screening. The comparison literature finds that O82580, Q9SQH7 and Q647G5 have been reported in the prior literature. Comparing with the previous literature, FFVPPSQQSPR of O82580 and FFVPPFQQSPR isopeptide fragment (which is marked in bold in Table 3) in Q9SQH7 have been reported, thereby verifying the feasibility of the screening method. In addition, 5 unreported specific peptide fragments (GRREEEEDEDEEEEGSNR, VSKEHVEELTK, KGSEEEGDITNPINLR, NNPFYFPSRR, EHVEELTK) in the allergen protein Ara h1(N1NG13) and unreported specific peptide fragments in proteins and subunits such as allergen proteins Ara h 8(B0YIU5), Ara h10 (Q647G5) are screened by the method.
TABLE 3 JIHUA 16 sensitized protein subunit and its specific peptide fragment
Ji flower 18(JH18) is high-oleic peanut, and as can be seen from Table 4, 76 sequences of 12 protein subunits in 9 peanut allergenic proteins meet the conditions after screening. The comparison literature finds that O82580, Q9SQH7, Q647G5 and Q45W87 have been reported in the prior literature. Comparing with the previous literature, ADEEEEYDEDEYEYDEEDR of O82580 and FFVPPFQQSPR isopeptide fragment (which is marked in bold in Table 4) in Q9SQH7 have been reported, thereby verifying the feasibility of the screening method. Besides, 5 unreported specific peptide fragments (GRREEEEDEDEEEEGSNR, VSKEHVEELTK, KGSEEEGDITNPINLR, NNPFYFPSRR, EHVEELTK) in the allergen proteins Ara h1(N1NG13) and unreported specific peptide fragments in proteins and subunits such as allergen proteins Ara h10 (Q647G5) and Ara h11 (Q45W87) are screened by the method.
TABLE 4 JI HUA 18 sensitized protein subunit and its specific peptide fragment
From the above examples, the present invention provides a method for detecting allergens in high oleic acid peanuts by using mass spectrometry, which comprises the following steps: carrying out mass spectrometry on the high oleic acid peanut extract by using a mass spectrometry parallel reaction monitoring method to obtain protein information; comparing and screening the protein information with bioinformatics data in a UniProt database to obtain allergic protein related to peanut allergy; screening out specific peptide fragments in the sensitized protein through specific screening conditions; the conditions for screening out specific peptide fragments include: 1) no modification is carried out; 2) the number of the spectrogram matched with the amino acid sequence is more than 3 in the atlas database; 3) the sequence is present only in one or both proteins; 4) the number of amino acids is 7-20; 5) the peptide fragment does not contain cysteine and methionine. The method provided by the invention can be used for qualitatively and quantitatively detecting the peanut allergenic protein, and is high in accuracy. The method can solve the defects of low flux, cross interference and the like of an immunological method, can also solve the defect that a PCR method cannot directly detect the allergenic protein, can also confirm the allergenic protein and the peptide segment, and specifically screen out the corresponding peptide segment, thereby laying a foundation for identifying the peanut allergen by using an isotope dilution method subsequently.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A method for detecting allergens in high-oleic-acid peanuts by utilizing a mass spectrometry method comprises the following steps:
carrying out mass spectrometry on the high oleic acid peanut extract by using a mass spectrometry parallel reaction monitoring method to obtain protein information;
comparing and screening the obtained protein information with bioinformatics data in a UniProt database to obtain allergic protein related to peanut allergy;
screening out specific peptide fragments in the sensitized protein through specific screening conditions to obtain specific peptide fragments in the specific allergen;
the conditions for screening out specific peptide fragments include: 1) no modification is carried out; 2) the number of the spectrogram matched with the amino acid sequence is more than 3 in the atlas database; 3) the sequence is present only in one or both proteins; 4) the number of amino acids is 7-20; 5) the peptide fragment does not contain cysteine and methionine.
2. The method of claim 1, wherein the results of the mass spectrometry analysis are retrieved using the Proteome Discover software in the UniProt/Swiss-prot database.
3. The method of claim 1, wherein the column conditions for mass spectrometry are: a C-18 chromatographic column; flow rate: 300 mu L/min; the sample introduction volume is 2 mL; solution A: 0.1% aqueous formic acid solution, solution B: 0.1% formic acid in acetonitrile, gradient elution;
mass spectrum conditions: electron source: ESI; the scanning mode is as follows: a PRM; adopting a positive ion mode, wherein the temperature of an ion transmission tube is 320 ℃; the primary full scanning range is 350-1550 m/z; the secondary scan AGC is set to 5.0e4, the ion implantation time IT is set to 100ms, and the collision energy is 32 eV.
4. The method of claim 1, wherein the high oleic peanut extract is produced by:
peeling peanut, removing red skin, pulverizing, and defatting to obtain defatted peanut powder;
mixing the defatted peanut powder with a buffer solution, shearing at 8500-9500 rpm for 55-65 s, magnetically stirring at 4 ℃ for 4-4.2 h, centrifuging the obtained peanut protein homogenate, and collecting a supernatant;
boiling the supernatant, loading, performing electrophoresis for 50min by using a Bio-rad precast gel and a vertical electrophoresis tank at constant voltage of 150V, and decoloring after dyeing by using Coomassie brilliant blue to obtain a gel strip;
cutting the gel strip, chopping, decoloring with acetonitrile and ammonium bicarbonate, reducing disulfide bonds with dithiothreitol, performing enzymolysis with trypsin, treating at 35-40 ℃ for 10-15 h, then stopping the enzymolysis reaction, extracting the obtained peptide segment for multiple times with a dissolving solution containing 0.1% of trifluoroacetic acid and 50% of acetone, concentrating the extracted peptide segment, re-dissolving in a formic acid solution, and centrifuging to obtain the high-oleic-acid peanut extract.
5. The method according to claim 4, wherein the molecular weight of the allergenic protein in the gel bands is between 5kDa and 75 kDa.
6. The method of claim 1, wherein the specific peptide fragment comprises one or more of Q9SQH7, B0YIU5, B6CG41, Q647G5, O82580, and Q45W 87.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110294700A1 (en) * | 2010-06-01 | 2011-12-01 | Thelen Jay J | High-throughput quantitation of crop seed proteins |
| CA2958074A1 (en) * | 2014-08-11 | 2016-02-18 | Dow Agrosciences Llc | Systems and methods for selective quantitation and detection of allergens |
| CN107290461A (en) * | 2017-07-14 | 2017-10-24 | 浙江工商大学 | A kind of method for the LC-MS analysis for setting up royal jelly allergic protein |
| CN109438551A (en) * | 2018-11-06 | 2019-03-08 | 北京蛋白世界生物科技有限公司 | A kind of method of trace protein sample concentration |
| CN109596729A (en) * | 2018-12-06 | 2019-04-09 | 中国农业科学院作物科学研究所 | Application of the construction method and soybean protein express spectra of soybean seed protein express spectra in analysis Biometabolic pathway |
-
2021
- 2021-04-19 CN CN202110433492.3A patent/CN113514574A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110294700A1 (en) * | 2010-06-01 | 2011-12-01 | Thelen Jay J | High-throughput quantitation of crop seed proteins |
| CA2958074A1 (en) * | 2014-08-11 | 2016-02-18 | Dow Agrosciences Llc | Systems and methods for selective quantitation and detection of allergens |
| CN107290461A (en) * | 2017-07-14 | 2017-10-24 | 浙江工商大学 | A kind of method for the LC-MS analysis for setting up royal jelly allergic protein |
| CN109438551A (en) * | 2018-11-06 | 2019-03-08 | 北京蛋白世界生物科技有限公司 | A kind of method of trace protein sample concentration |
| CN109596729A (en) * | 2018-12-06 | 2019-04-09 | 中国农业科学院作物科学研究所 | Application of the construction method and soybean protein express spectra of soybean seed protein express spectra in analysis Biometabolic pathway |
Non-Patent Citations (6)
| Title |
|---|
| 宁亚维等: "超高效液相色谱-串联质谱法检测食品中鸡蛋过敏原卵白蛋白", 《食品科学》 * |
| 洪宇伟等: "花生过敏原及其检测方法研究进展", 《食品安全质量检测学报》 * |
| 洪宇伟等: "超高效液相色谱-电喷雾质谱法检测花生过敏原Ara h 2", 《食品安全质量检测学报》 * |
| 蒋易蓉等: "靶向蛋白质组学质谱技术在食物过敏原定量检测中的应用", 《中国食品学报》 * |
| 詹丽娜等: "超高效液相色谱-四极杆/静电场轨道阱高分辨质谱检测食品中的牛奶过敏原酪蛋白", 《色谱》 * |
| 高梦笛等: "基于NanoLC-Orbitrap技术测定六种不同栽培品种大豆中多肽Lunasin含量", 《现代食品科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114755360A (en) * | 2022-06-15 | 2022-07-15 | 中国农业科学院蜜蜂研究所 | Method for evaluating sensitization of cabbage type rape bee pollen |
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