CN104569124A - Egg mass spectrometric detection signature sequence group and detection kit - Google Patents
Egg mass spectrometric detection signature sequence group and detection kit Download PDFInfo
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- CN104569124A CN104569124A CN201410235231.0A CN201410235231A CN104569124A CN 104569124 A CN104569124 A CN 104569124A CN 201410235231 A CN201410235231 A CN 201410235231A CN 104569124 A CN104569124 A CN 104569124A
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Abstract
The invention relates to an egg mass spectrometric detection signature sequence group and a detection kit, belonging to the field of the food detection. The invention relates to an egg mass spectrometric detection signature group as well as an egg allergen detection reagent and an egg allergen detection method. The egg mass spectrometric detection signature sequence group is high in sensitivity, good in precision, suitable for detecting the egg in a processed product containing the egg such as bread and ice cream and identifying the authenticity of the egg and convenient for being popularized in various detection institutions.
Description
Technical field
The invention provides a kind of detection method of egg, belong to field of food detection, specifically, relate to the technology detecting characteristic sequence group in egg with mass spectrometry method.
Background technology
Phagopyrism is the health problem be more and more taken seriously, and egg belongs to one of eight large food allergens.The allergic reaction of egg induction is different, has plenty of slight local reaction as oral allergy syndrome, has plenty of the reaction of serious systemic anaphylaxis, some or even fatal and intimate fatal allergic reaction.Due to the difference of US and European egg consumption figure, cause the susceptibility of anaphylactogen different.In Europe, egg allergy is very general.Comprise egg in 14 kinds of anaphylactogens that must mark that European Union requires in 2007/68/EC, in same Canadian CODEX STAN118-1979, equally clear and definite requirement has been made to the mark of anaphylactogen.In order to the requirement of satisfied supervision and the demand of allergic human population, need to develop corresponding detection method.The detection method of current egg has euzymelinked immunosorbent assay (ELISA), but egg allergen and birch factor Ⅰ have the anaphylactogen of cross reaction, therefore easily cause false positive results.
Summary of the invention
The technical issues that need to address of the present invention are to provide the egg characteristic sequence for Mass Spectrometer Method.
The present invention is achieved by the following technical solutions:
1. egg allergen Ovalbumin amino acid sequence
2. the screening of characteristic sequence: find applicable amino acid sequence as tags detected for egg allergen Ovalbumin by PinPoint software.
Egg allergen Ovalbumin amino acid sequence Uniprot:P01012.
Confirm that the egg Mass Spectrometer Method set of tags be applicable to is through screening:
1. YPILPEYLQCVK
2. HIATNAVLFFGR
Table 1 egg allergen protein characteristic peptide section and SRM ion pair
Egg of the present invention detects Reagent Kit, it is characterized in that, it is the reagent for being detected egg allergen by mass spectroscopy, the egg Mass Spectrometer Method characteristic sequence group containing the invention described above.
The detection method of a kind of egg allergen of the present invention, it is characterized in that, it is for preparing the method for egg Mass Spectrometer Method characteristic sequence group by the method for enzymolysis, containing following step: extract the protein in sample with the Extraction buffer in kit, after modifying, carried out the preparation of egg Mass Spectrometer Method characteristic sequence group by the method for enzymolysis, be dissolved in dissolving damping fluid, mass spectrometer carries out ionization and detection.
Sample can be the product that bread, cake, ice cream, biscuit etc. may contain egg, judges whether it has egg ingredients by detecting egg Mass Spectrometer Method characteristic sequence group.If egg Mass Spectrometer Method characteristic sequence group testing result is positive simultaneously, then contain egg ingredients in sample, if egg allergen label testing result is negative, then do not contain egg ingredients in sample.
In addition, according to conventional methods, this egg tags detected can be applied to the mass spectrometer of different instrument company.According to general knowledge, the volume of each component can change according to condition difference, and the best of breed of each different batches reagent all may be different.
3. detection method
Trace routine: (1) gets testing sample 2 g, adds Extraction buffer 10ml, 60 DEG C of centrifugal 10min of vibration 3h, 14000g, get supernatant;
(2) add solution 1M DTT 1 μ l, 37 degree of 2h, then add 1M IAA 5 μ l room temperature 40min.
(3) after reaction, directly add 2 μ L trypsin (200 ng/ μ L), and to vibrate enzymolysis 1.5 hours in 37 ° of C.
(4) after enzymolysis completes, in 20, centrifugal 5 minutes of 000 g, gets supernatant, direct injection analysis.
(5) liquid phase chromatogram condition:
Chromatographic column: Hypersil GOLD aQ post (100 x 2.1 mm, 1.9 μm); Applied sample amount: 10 μ L; A phase: 0.1% aqueous formic acid; B phase: 0.1% formic acid acetonitrile solution; Flow velocity: 300 μ L/min; Analyze gradient: 0 – 2 min 5% B, 2 – 12 min 5% – 95% B, 12 – 17 min 95% B, 17 – 18 min, 95% – 5% B, 18 – 23 min 5% B.
(6) tandem mass spectrum condition:
Scan pattern: positive ion, SRM; Spray voltage: 3000 V; Gasification temperature: 300 ° of C sheath air pressure: 30 psi; Assist gas pressure (arbitrary units): 10; Ion transfer tube temperature: 350 ° of C; Collision gas (Ar): 1.5 mTorr; Q1/Q3 resolution (FWHM): 0.7; Residence time: 0.04 s; SRM scan ion to and collision energy 26.
(7) egg allergen protein sequence is from Swiss-Prot database; The selection of selectivity peptide section and characteristic fragment and collision energy optimization are analyzed by PinPoint software (Thermo), obtain in conjunction with LTQ-Orbitrap high resolution mass spectrum (Thermo) full scan confirmation; Mass spectrometric data carries out qualitative analysis by Xcalibur Qual Browser software (Thermo), carries out quantitative test by Xcalibur Quan Browser software (Thermo).
(8) linear regression processing is carried out with peak area and Mass Spectrometer Method characteristic sequence group concentration, the concentration that has that it's too late of the egg allergen in calculation sample.
Should be understood that, the separation vessel that method is used can be carbon 18 post, also can be the serial connection of ion exchange column and carbon 18 post or two dimension one scapus of ion exchange column and carbon 18 post.Its detection method can be Mass Spectrometry detection method, can be triple level Four bars, or compound electric field track trap cyclotron resonance mass spectrum, or tandem mass spectrum.
Enzyme can be pancreatin, also can be that other have the enzyme of identical function.
Second object of the present invention is to provide the detection kit according to above-mentioned detection method, comprising:
(1) Extraction buffer 0.2M Tris-HCl pH8.1 ~ 8.3; With
(2) pancreatin 10ng/ μ l, solvent is 50mM ammonium hydrogencarbonate; With
(3) DTT 1M, solvent is 100mM ammonium hydrogencarbonate; With
(4) IAA 1M, solvent is 100mM ammonium hydrogencarbonate;
(5) the mark peptide of Mass Spectrometer Method characteristic sequence group.
Compared with prior art, the present invention relates to and has the following advantages:
1. Mass Spectrometer Method characteristic sequence group of the present invention can identify egg allergen 11S globulin specifically;
2. can carry out egg allergen testing at existing Mass Spectrum Laboratory, not need to add any equipment, a large amount of examination and confirmation requirements of one's work can be met;
3., when using tags detected of the present invention, effectively can shorten detection time;
4. owing to having used two tags detected in method simultaneously, the needs of qualitative detection can be met, the needs quantitatively detected can also be met simultaneously;
5. the scope of application is more extensive, can not only carry out anaphylactogen discriminating, and can detect deep-processed food;
6. this method can be applicable to the True-false distinguish of egg.
In a word, Mass Spectrometer Method characteristic sequence group of the present invention, Extraction buffer and use the preparation method of their tags detected, can detect egg rapidly and simply, be very effective in inspection and quarantine system etc.
Embodiment
Only further describe the present invention by the mode of reference nonrestrictive embodiment below now.But should be appreciated that the following examples are only illustratively, should by any way when doing the restriction overall to the invention described above.Unless otherwise noted, embodiments of the invention use the mass-spectrometric technique in this area.These technology are known by the technical staff, and have detailed explanation in the literature.
Embodiment 1
Sample: in the packaging of bread, mark statement is wherein containing egg ingredients, detects by mass spectrographic method.
Get bread matrix 2 g, add Extraction buffer 10ml, 60 DEG C of centrifugal 10min of vibration 3h, 14000g, get supernatant, add solution 1M DTT 1 μ l, 37 degree of 2h, then add 1M IAA 5 μ l room temperature 40min.After reaction, directly add 2 μ L trypsin (200 ng/ μ L), and to vibrate enzymolysis 1.5 hours in 37 ° of C.
After enzymolysis completes, in 20, centrifugal 5 minutes of 000 g, gets supernatant, direct injection analysis.
Liquid phase chromatogram condition:
Chromatographic column: Hypersil GOLD aQ post (100 x 2.1 mm, 1.9 μm); Applied sample amount: 10 μ L; A phase: 0.1% aqueous formic acid; B phase: 0.1% formic acid acetonitrile solution; Flow velocity: 300 μ L/min; Analyze gradient: 0 – 2 min 5% B, 2 – 12 min 5% – 95% B, 12 – 17 min 95% B, 17 – 18 min, 95% – 5% B, 18 – 23 min 5% B.
Tandem mass spectrum condition:
Scan pattern: positive ion, SRM; Spray voltage: 3000 V; Gasification temperature: 300 ° of C sheath air pressure: 30 psi; Assist gas pressure (arbitrary units): 10; Ion transfer tube temperature: 350 ° of C; Collision gas (Ar): 1.5 mTorr; Q1/Q3 resolution (FWHM): 0.7; Residence time: 0.04 s; SRM scan ion to and collision energy 26.
Egg allergen protein sequence is from Swiss-Prot database; The selection of selectivity peptide section and characteristic fragment and collision energy optimization are analyzed by PinPoint software (Thermo), obtain in conjunction with LTQ-Orbitrap high resolution mass spectrum (Thermo) full scan confirmation; Mass spectrometric data carries out qualitative analysis by Xcalibur Qual Browser software (Thermo), carries out quantitative test by Xcalibur Quan Browser software (Thermo).The tags detected used during detection is: YPILPEYLQCVK and HIATNAVLFFGR.
<110> Animal-Plant and food Detecting Center, Tianjin Exit-Entery Inspection & Quarant
<120> egg Mass Spectrometer Method characteristic sequence group and detection kit
<130> 20140731
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213> artificial sequence
<400> 1
Tyr Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys
1 5 10
<210> 2
<211> 12
<212> PRT
<213> artificial sequence
<400> 2
His Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg
1 5 10
Claims (8)
1. an egg Mass Spectrometer Method characteristic sequence group, is characterized in that, it is the tags detected group for being detected egg allergen by mass spectroscopy, containing following characteristics sequence:
Characteristic sequence 1:YPILPEYLQCVK
Characteristic sequence 2:HIATNAVLFFGR.
2. egg Mass Spectrometer Method characteristic sequence group according to claim 2, the converted products that described sample can be bread, cake, ice cream, biscuit contain egg.
3. the Mass Spectrometer Method characteristic sequence group described in claim 1 to 2 needs, with egg ingredients detection kit process sample, to it is characterized in that comprising:
(1) Extraction buffer 0.2M Tris-HCl pH8.1 ~ 8.3; With
(2) pancreatin 10ng/ μ l, solvent is 50mM ammonium hydrogencarbonate; With
(3) the mark peptide of Mass Spectrometer Method characteristic sequence group.
4. a kind of detection method of egg in claims 1 to 3, wherein step comprises: extract the protein in sample with the Extraction buffer in kit, carry out enzymolysis, be dissolved in dissolving damping fluid, by separation vessel isolated peptides section, mass spectrometer carries out ionization and detection, carries out linear regression processing with peak area and label concentration, the concentration that has that it's too late of the egg allergen in calculation sample.
5. the detection method of egg Mass Spectrometer Method characteristic sequence group according to claim 4, it is in described step, the separation vessel used can be carbon 18 post, also can be the serial connection of ion exchange column and carbon 18 post or two dimension one scapus of ion exchange column and carbon 18 post.
6. the detecting device of egg Mass Spectrometer Method characteristic sequence group according to claim 4 can be triple level Four bars, or compound electric field track trap cyclotron resonance mass spectrum, or tandem mass spectrum.
7. a determination methods for testing result, is characterized in that, if there is the positive findings of egg Mass Spectrometer Method characteristic sequence group simultaneously, containing egg in sample, does not contain egg ingredients if there is in egg Mass Spectrometer Method characteristic sequence group negative findings then sample.
8. a determination methods for testing result, is characterized in that, quantitatively can be detected by the method for linear regression to the egg content in sample.
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| CN201410235231.0A CN104569124A (en) | 2014-05-29 | 2014-05-29 | Egg mass spectrometric detection signature sequence group and detection kit |
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| CN201410235231.0A CN104569124A (en) | 2014-05-29 | 2014-05-29 | Egg mass spectrometric detection signature sequence group and detection kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107056932A (en) * | 2016-12-24 | 2017-08-18 | 深圳大学 | A kind of amino acid sequence, fusion protein of the epitope containing OVA and its preparation method and application |
| CN109557193A (en) * | 2018-07-09 | 2019-04-02 | 中国检验检疫科学研究院 | A kind of mass spectrometry detection method of sesame main allergen |
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| WO2003046563A1 (en) * | 2001-11-27 | 2003-06-05 | Yorktest Laboratories Limited | Food intolerance immunoassay |
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| CN101893636A (en) * | 2010-06-24 | 2010-11-24 | 江南大学 | Enzyme-linked immunosorbent assay method for egg allergen ovalbumin in foods |
| CN102331495A (en) * | 2011-06-21 | 2012-01-25 | 沃克(天津)生物科技有限公司 | Food allergen assay kit and preparation method thereof |
| CN202710568U (en) * | 2012-06-15 | 2013-01-30 | 北京中检葆泰生物技术有限公司 | Colloidal gold test paper strip and kit both capable of rapidly detecting egg allergen ovalbumin |
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2014
- 2014-05-29 CN CN201410235231.0A patent/CN104569124A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003046563A1 (en) * | 2001-11-27 | 2003-06-05 | Yorktest Laboratories Limited | Food intolerance immunoassay |
| JP2007278830A (en) * | 2006-04-06 | 2007-10-25 | Ulvac Japan Ltd | Device and method for detecting food components |
| CN101893636A (en) * | 2010-06-24 | 2010-11-24 | 江南大学 | Enzyme-linked immunosorbent assay method for egg allergen ovalbumin in foods |
| CN102331495A (en) * | 2011-06-21 | 2012-01-25 | 沃克(天津)生物科技有限公司 | Food allergen assay kit and preparation method thereof |
| CN202710568U (en) * | 2012-06-15 | 2013-01-30 | 北京中检葆泰生物技术有限公司 | Colloidal gold test paper strip and kit both capable of rapidly detecting egg allergen ovalbumin |
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| J. HEICK 等: "First screening method for the simultaneous detection of seven allergens by liquid chromatography mass spectrometry", 《JOURNAL OF CHROMATOGRAPHY A》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107056932A (en) * | 2016-12-24 | 2017-08-18 | 深圳大学 | A kind of amino acid sequence, fusion protein of the epitope containing OVA and its preparation method and application |
| CN107056932B (en) * | 2016-12-24 | 2021-04-09 | 深圳大学 | Amino acid sequence, fusion protein containing OVA epitope, and preparation method and application thereof |
| CN109557193A (en) * | 2018-07-09 | 2019-04-02 | 中国检验检疫科学研究院 | A kind of mass spectrometry detection method of sesame main allergen |
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