CN104569125A - Hazel mass spectrometric detection signature sequence group and detection kit - Google Patents
Hazel mass spectrometric detection signature sequence group and detection kit Download PDFInfo
- Publication number
- CN104569125A CN104569125A CN201410235992.6A CN201410235992A CN104569125A CN 104569125 A CN104569125 A CN 104569125A CN 201410235992 A CN201410235992 A CN 201410235992A CN 104569125 A CN104569125 A CN 104569125A
- Authority
- CN
- China
- Prior art keywords
- fibert
- sequence group
- hazel
- mass spectrometer
- characteristic sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention relates to a hazel mass spectrometric detection signature sequence group and a detection kit, and particularly relates to a reagent for detecting hazel allergen by virtue of a mass spectrometry, a hazel mass spectrometric detection signature sequence group comprising the reagent and a method for preparing the hazel mass spectrometric detection signature sequence group in an enzymolysis method. The method comprises the following steps: extracting proteins in a test sample by utilizing an extraction buffer solution in the kit, preparing the hazel mass spectrometric detection signature sequence group by virtue of the enzymolysis method after the protein is decorated, dissolving the prepared hazel mass spectrometric detection signature sequence group in a dissolving buffer solution, and carrying out the ionization and detection on a mass spectrometer. The test sample can be a product containing hazel such as bread, cake, ice cream and biscuits. Whether the hazel ingredient exists or not can be determined by detecting the hazel mass spectrometric detection signature sequence group. If a hazel mass spectrometric detection signature sequence group detection result is positive, the hazel ingredient is contained in the test sample; if the hazel allergen tag detection result is negative, the test sample does not contain the hazel ingredient.
Description
Technical field
The invention provides a kind of detection method of fibert, belong to field of food detection, specifically, relate to the technology detecting characteristic sequence group in fibert with mass spectrometry method.
Background technology
Phagopyrism is the health problem be more and more taken seriously, and nut belongs to one of eight large food allergens, relates to 90% of phagopyrism.Tree nut allergen is stable for food processing and digestion.In some seeds, similar stable anaphylactogen is determined, because their unique taste, seed and nut are through being usually used in food industry.Since the nineties, due to the variation of food product, the recipe relevant to nut and seed has been caused to get more and more.Perhaps, the product not comprising nut or seed can utilize the equipment of processed nut and seed, defines potential risks cross-contact.For the exploration discovery containing anaphylactogen in the product enriching nut or seed compositions of current hypothesis, these anaphylactogens have caused some serious allergic reactions.This shows, the method detecting nut and Seed Allergens is the important means that food industry controls manufacture and handling procedure.Because fibert make use of a very long time, especially in West Europe, many effort are the detections devoting fibert.
1996, Tariq etc. delivered one section of investigation about popular tree nut allergies disease, comprising 1218 neonates of the Isle of Wight of Britain.Through dermoreaction test, wherein 1.2% is that tree nut Ig E is positive, clinical response 0.16%.The research of this people such as with Sicherer is consistent, utilize random digit dialing telephone poll more than 12 000 the individuality in the U.S., evaluate tree nut allergies disease patient 0.5%.The allergic reaction of nut 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 nut consumption figure, cause the susceptibility of anaphylactogen different.In Europe, fibert allergy is very general, and often combine with birch pollen allergy, and cause primarily of English walnut in U.S.'s allergic reaction, cashew nut, almond, hickory nut, American pistachios take second place.Comprise fibert 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 fibert has euzymelinked immunosorbent assay (ELISA), but fibert anaphylactogen 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 fibert characteristic sequence for Mass Spectrometer Method.
The present invention is achieved by the following technical solutions:
1. fibert anaphylactogen 11S immunoglobulin amino acid sequence
2. the screening of characteristic sequence: find applicable amino acid sequence as tags detected for fibert anaphylactogen 11S globulin by PinPoint software.
Fibert anaphylactogen 11S immunoglobulin amino acid sequence GenBank:AAL73404.1
1 maklilvsfs lcllvlfngc lginvglrrq qqryfgecnl drlnaleptn rieaeacqie
61 swdhndqqfq cagvavirrt iepnglllpq ysnapeliyi ergrgitgvl fpgcpetfed
121 pqqqsqqgqr qgqgqsqrse qdrhqkirhf regdiialpa gvahwcyndg dspvvtvsll
181 htnnyanqld enprhfylag npddehqrqg qqqfgqrrrq qqhshgeqge qeqqgegnnv
241 fsgfdaefla dafnvdvdta rrlqsnqdkr rnivkvegrl qvvrpersrq ewerqerqer
301 eseqererqr rqggrgrdvn gfeeticslr lrenictrsr adiyteqvgr intvnsntlp
361 vlrwlqlsae rgdlqregly vphwnlnahs vvyairgrar vqvvddngnt vfddelrqgq
421 vltipqnfav akraesegfe wvafktndna qisplagrts airalpddvl anafqisree
481 arrlkynrqe ttlvrssrss serkrrsese graea
Confirm that the fibert Mass Spectrometer Method set of tags be applicable to is through screening:
1.INTVNSNTLPVLR
2.ADIYTEQVGR
Table 1 fibert allergen protein characteristic peptide section and SRM ion pair
Fibert of the present invention detects Reagent Kit, it is characterized in that, it is the reagent for being detected fibert anaphylactogen by mass spectroscopy, the fibert Mass Spectrometer Method characteristic sequence group containing the invention described above.
The detection method of a kind of fibert anaphylactogen of the present invention, it is characterized in that, it is for preparing the method for fibert 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 fibert 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 fibert, judges whether it has fibert composition by detecting fibert Mass Spectrometer Method characteristic sequence group.If fibert Mass Spectrometer Method characteristic sequence group testing result is positive simultaneously, then contain fibert composition in sample, if fibert anaphylactogen label testing result is negative, then do not contain fibert composition in sample.
In addition, according to conventional methods, this fibert 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 in table 1.
(7) fibert 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 fibert anaphylactogen 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 fibert anaphylactogen 11S globulin specifically;
2. can carry out the work of fibert Allergic skin test 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 fibert.
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 fibert rapidly and simply, be very effective in inspection and quarantine system etc.
Accompanying drawing explanation
Fig. 1 fibert anaphylactogen 11S globulin A DIYTEQVGR peptide section MS/MS spectrogram.
Fig. 2 fibert anaphylactogen 11s globulin INTVNSNTLPVLR peptide section mass spectrogram.
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 fibert composition, 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 in table 1.
Fibert 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: 1.INTVNSNTLPVLR and 2.ADIYTEQVGR.
Claims (8)
1. a fibert Mass Spectrometer Method characteristic sequence group, is characterized in that, it is the tags detected group for being detected fibert anaphylactogen by mass spectroscopy, containing following characteristics sequence:
Characteristic sequence 1:INTVNSNTLPVLR
Characteristic sequence 2:ADIYTEQVGR.
2. fibert Mass Spectrometer Method characteristic sequence group according to claim 2, the converted products that described sample can be bread, cake, ice cream, biscuit contain fibert.
3. the Mass Spectrometer Method characteristic sequence group described in claim 1 to 2 needs, with fibert composition 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 fibert 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 fibert anaphylactogen in calculation sample.
5. the detection method of fibert 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 fibert 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 fibert Mass Spectrometer Method characteristic sequence group simultaneously, containing fibert in sample, does not contain fibert composition if there is in fibert 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 fibert content in sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410235992.6A CN104569125A (en) | 2014-05-29 | 2014-05-29 | Hazel mass spectrometric detection signature sequence group and detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410235992.6A CN104569125A (en) | 2014-05-29 | 2014-05-29 | Hazel mass spectrometric detection signature sequence group and detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104569125A true CN104569125A (en) | 2015-04-29 |
Family
ID=53085687
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410235992.6A Pending CN104569125A (en) | 2014-05-29 | 2014-05-29 | Hazel mass spectrometric detection signature sequence group and detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104569125A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107142330A (en) * | 2017-07-11 | 2017-09-08 | 中国林业科学研究院林业研究所 | The method that hazel Germplasm Identification is carried out using core ITS sequence |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101109750A (en) * | 2007-07-27 | 2008-01-23 | 杭州浙大生科生物技术有限公司 | Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof |
| CN101387643A (en) * | 2008-10-20 | 2009-03-18 | 杭州浙大生物基因工程有限公司 | Multichannel allergen rapid detection kit and method for making same |
| WO2010114912A1 (en) * | 2009-03-31 | 2010-10-07 | University Of North Carolina At Greensboro | Minimally invasive assessment of ige mediated allergy |
| US20110294700A1 (en) * | 2010-06-01 | 2011-12-01 | Thelen Jay J | High-throughput quantitation of crop seed proteins |
-
2014
- 2014-05-29 CN CN201410235992.6A patent/CN104569125A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101109750A (en) * | 2007-07-27 | 2008-01-23 | 杭州浙大生科生物技术有限公司 | Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof |
| CN101387643A (en) * | 2008-10-20 | 2009-03-18 | 杭州浙大生物基因工程有限公司 | Multichannel allergen rapid detection kit and method for making same |
| WO2010114912A1 (en) * | 2009-03-31 | 2010-10-07 | University Of North Carolina At Greensboro | Minimally invasive assessment of ige mediated allergy |
| US20110294700A1 (en) * | 2010-06-01 | 2011-12-01 | Thelen Jay J | High-throughput quantitation of crop seed proteins |
Non-Patent Citations (1)
| Title |
|---|
| J. HEICK 等: "First screening method for the simultaneous detection of seven allergens by liquid chromatography mass spectrometry", 《JOURNAL OF CHROMATOGRAPHY A》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107142330A (en) * | 2017-07-11 | 2017-09-08 | 中国林业科学研究院林业研究所 | The method that hazel Germplasm Identification is carried out using core ITS sequence |
| CN107142330B (en) * | 2017-07-11 | 2020-06-02 | 中国林业科学研究院林业研究所 | Method for carrying out hazel germplasm identification by using nuclear ITS sequence |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109085278B (en) | Kit for simultaneously detecting multiple amino acids by liquid chromatography-tandem mass spectrometry and application thereof | |
| CN105067698B (en) | Quantify insulin like growth factor-1 and Insulin-like growth factor-II with high resolution mass spec method | |
| CN113480599A (en) | Characteristic polypeptide for identifying deer antler glue of sika deer or red deer and application thereof | |
| CN106589063A (en) | Donkey-source characteristic peptide group and application process thereof in qualitative detection of donkey skin and donkey-hide gelatin | |
| CN102435680B (en) | Bovine serum albumin non-labeled mass spectrum qualitative and quantitative detection method | |
| CN109791131A (en) | LC/MS/MS analysis for the identification of the meat kind of raw meat and meat processing product | |
| CN108291916A (en) | Mass spectrography detects amyloid-beta | |
| CN106589114A (en) | Porcine-derived characteristic peptide and application processes thereof in pigskin and colla corii asini qualitative detection | |
| CN114113381B (en) | Syngnathus schutz characteristic polypeptide, application thereof and method for identifying comfortable Syngnathus schutz | |
| CN106053697B (en) | A kind of ox source property characteristic polypeptide and its application | |
| CN110678756B (en) | Method for absolute quantification of low abundance polypeptides using mass spectrometry | |
| CN104569243A (en) | Peanut mass spectrometric detection signature sequence group and detection kit | |
| CN106198790A (en) | A kind of horse and mule common characteristic polypeptide and application thereof | |
| CN107153103B (en) | Method for determining contents of various mycotoxins in fresh milk sample | |
| CN104561256A (en) | Walnut mass spectrometric detection characteristic sequence group and detection kit | |
| CN104569124A (en) | Egg mass spectrometric detection signature sequence group and detection kit | |
| CN104569125A (en) | Hazel mass spectrometric detection signature sequence group and detection kit | |
| Yang et al. | Development of an SI-traceable HPLC–isotope dilution mass spectrometry method to quantify β-Lactoglobulin in milk powders | |
| CN104569171A (en) | Almond mass spectrometric detection characteristic sequence group and detection kit | |
| CN115494182A (en) | Method and kit for detecting 18 antibiotics in blood by liquid chromatography-tandem mass spectrometry | |
| JP7438224B2 (en) | Fast sample workflow for HbA1c measurement based on LC-MS | |
| CN114276431A (en) | Camel milk characteristic peptide segment combination and identification method | |
| CN113820424A (en) | HPLC-MS/MS method for simultaneously determining concentration of 14 antidepressants in human plasma | |
| CN113009050A (en) | Detection method and application of quinolone compounds in dairy products | |
| CN112255327A (en) | Method for detecting glufosinate content in dairy product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150429 |