CN109557228A - A method of identifying bird's nest and its adulterant using signature peptide fragment - Google Patents
A method of identifying bird's nest and its adulterant using signature peptide fragment Download PDFInfo
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- CN109557228A CN109557228A CN201711374365.0A CN201711374365A CN109557228A CN 109557228 A CN109557228 A CN 109557228A CN 201711374365 A CN201711374365 A CN 201711374365A CN 109557228 A CN109557228 A CN 109557228A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
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Abstract
The present invention relates to bird's nest and its MRM qualitative checking methods of four kinds of common adulterant (pigskin, tremella, egg white and air bladder) ingredients.This method passes through the protein in the extraction with aqueous solution bird's nest and its common adulterant of urea containing 7M and 2M thiocarbamide, digestion is stayed overnight through trypsase, with nanoliter liquid chromatogram high-resolution tandem mass spectrum (NanoLC-Q-TOF) analysis after peptide fragment desalination, the data that high resolution mass spectrum obtains are carried out using 5.0 software of Proteinpilot to search library identification, it is high to choose response, no leakage is cut, without modification, the peptide fragment of length < 25 amino acid carries out Blast comparison on NCBI, excavation obtains the specific peptide fragment of bird's nest and its common adulterant, MRM ion pair is constructed using specific peptide fragment of the SKYLINE software to bird's nest and its common adulterant simultaneously.This method is easy to operate, and accuracy is high, and stability is good, and primary experiment can identify bird's nest simultaneously and its mix pseudo- ingredient (one or more of pigskin, tremella, egg white and air bladder), consumes normal market order for maintenance bird's nest and provides technical support.
Description
Technical field
The present invention relates to the applications for identifying field in the bird's nest true and false based on mass spectrographic proteomic techniques, mainly include bird's nest
And its specific mark peptide fragment of adulterant (pigskin, tremella, egg white and air bladder) excavation and in the triple level four bars of liquid chromatogram
The foundation of multiple-reaction monitoring (MRM) method on tandem mass spectrum.
Background technique
Bird's nest --- a kind of excellent tonic product that the distinctive medicine food in Southeast Asia is all suitable, is Apodidae aerodramus gold not of the same race
The nest that silk swallow (based on Java esculent swift and big esculent swift) is built in the cave of island with saliva, for breeding offspring.Bird's nest it
So being eaten by the mankind, it initially may be only taken as a kind of food that can be allayed one's hunger, but not with human civilization
Disconnected development, bird's nest enriching yin are supported the unique medicinal efficacies such as lung, preventing phlegm from forming and stopping coughing and are found at leisure, then eat bird's nest and become in China
It is of long standing and well established at a kind of culture of health preserving that can be passed on.
China is the main body entrance state of bird's nest, is restored from 2014 to Malaysia and Indonesian bird's nest trade
Afterwards, 39.3 tons in 2016 have been risen to from 3.091 tons of 2014 according to the import volume of port statistical data bird's nest.Bird's nest demand
Continuous increase of amount imparts the huge profit margin of bird's nest industry with the high of the market price, and illegal retailer is in great number benefit
It makes a desperate move under the driving of profit, true bird's nest is pretended to be with gluing, dyeing, adulterated bird's nest, high price bird's nest, swallow are pretended to be with low price bird's nest
The processing link and intermediate links of the phenomenon that nest is faked long-term existence bird's nest.For specification bird's nest market, consumer's interests are protected,
The research work that the bird's nest true and false identifies is underway always from the nineties in last century, and false distinguishing method is also constantly being weeded out the old and bring forth the new.
Gradually turned by the method for the identification of dependence character, microscope or stereoscopic sem observation and traditional Physico-chemical tests currently, the bird's nest true and false identifies
The method for becoming relying on large-scale instrument, including bird's nest false distinguishing method (ultraviolet-visible spectrophotometry, efficient liquid based on sialic acid
Phase chromatography, efficient liquid phase tandem mass spectrometry and the chromatography of ions), bird's nest false distinguishing method based on protein (enzyme-linked immunization,
Capillary electrophoresis and dielectrophoresis method), the bird's nest false distinguishing method based on amino acid, based on DNA bird's nest false distinguishing method (in real time
Fluorescent PCR method and DNA bar code method) and bird's nest spectrum false distinguishing method (ultraviolet spectroscopy, infra-red sepectrometry, Raman spectroscopy and height
Light spectrum image-forming method) etc..The emphasis of these methods exploitation is different and has limitation, such as the enzyme linked immunological of sialoglycoprotein
Although method can judge the true and false of bird's nest from the variation of sialoglycoprotein content, adulterant identity cannot be judged；Saliva
The artificial synthesized content for causing sialic acid in bird's nest of acid is not suitable anymore for as judging the feature of the bird's nest true and false；For another example infrared spectroscopy
Method is although lossless, quick, but to the skill requirement of testing staff it is higher be not easy it is universal etc..It is found by document, in bird's nest
Common adulterant mainly has pigskin, tremella, egg white and air bladder, therefore needs to develop a kind of high sensitivity, favorable reproducibility, high pass
The distinguishing method between true and false of common adulterant in amount, the bird's nest easily promoted.
The excavation of specific peptide segment mark object is one of classic applications of proteomic techniques, can be not only used to qualitative
It can also be used to precisely quantitative.MRM(multiple-reaction monitoring) it is that one kind based on triple quadrupole rods tandem mass spectrometries is with higher sensitivity
Scanning mode is targeted, enhanced daughter ion scanning can with data dependence be triggered while MRM scanning, it is fixed to greatly reduce
Property detection false positive rate, improve the accuracy of qualification result.
Summary of the invention
It is a kind of based on mass spectrum proteomic techniques high sensitivity, favorable reproducibility, high throughput, easily popularization the present invention is directed to establish
Bird's nest in common adulterant pigskin, tremella, egg white and air bladder discrimination method.
A kind of distinguishing method between true and false of the bird's nest based on mass spectrum proteomic techniques includes the following steps:
Sample pre-treatments: bird's nest and its common adulterant pigskin, tremella and air bladder sample pass through liquid nitrogen grinding Cheng Fenhou -40
DEG C save；- 40 DEG C of preservations after egg white packing.
Protein Extraction and its assay: with the extraction with aqueous solution bird's nest of urea containing 7M and 2M thiocarbamide and its common
Protein in adulterant pigskin, tremella, egg white and air bladder, -20 DEG C of protein extract solution preservations.With bovine serum albumin(BSA)
(BSA) protein content in solution is measured for protein standard substance Bradford method.
Protein digestion and high-resolution Tandem Mass Spectrometry Analysis: the protein extract solution containing 200 μ g albumen is taken, through two
After sulphur threitol (DTT) reduction, iodoacetamide (IAA) alkylation, trypsase stays overnight digestion, and high-resolution is connected after peptide fragment desalination
Mass spectrum (NanoLC-Q-TOF) analysis.
Using 5.0 software of Proteinpilot to the data that high resolution mass spectrum obtains search library identification bird's nest and its
Protein in common adulterant is chosen response height in the peptide fragment identified, is cut without leakage, without modification, length < 25 amino acid
Peptide fragment Blast comparison, the bird's nest excavated and its common adulterant pigskin, tremella, egg white and air bladder are carried out on NCBI
Specific peptide segment mark object it is as follows:
The foundation of bird's nest and its common adulterant MRM qualitative checking method: according to bird's nest and its high-resolution of common adulterant
Mass Spectrometric Identification result (Proteinpilot 5.0) and SKYLINE software are to bird's nest and its common adulterant pigskin, tremella, egg white
The MRM ion pair constructed with the specific peptide segment mark object of air bladder, and optimize ion pair impact energy (CE value) with 2 for step-length.
The distinguishing method between true and false of bird's nest of the present invention based on mass spectrum proteomic techniques, wherein stepIt is described
High-resolution tandem mass spectrum (NanoLC-Q-TOF) analysis condition it is as follows:
NanoLC-MS/MS high-resolution tandem mass spectrum (Eksigent 400 and TripleTOFTM 5600), Q1-IDA-EPI scanning
Mode.Eksigent chip chromatographic column (ChromXP C18-CL, 75 μ m, 15 cm, 3 μm of partial sizes, 120), mobile phase A:
2% acetonitrile, 0.1% formic acid, 98% water；Mobile phase B: 98% acetonitrile, 0.1% formic acid, 2% water；0.3 μ L/min of flow velocity, sample introduction body
4 μ L of product, mobile phase gradient such as table 1:
The gradient elution program of 1 NanoLC of table
Loading Pump flow velocity 2 μ L/min, 10min.ESI ion source, positive ion mode, Ion spray voltage:
2.4 kv；GS1:6；Curtain gas:30；DP:100；Q1 scanning range: 350-1250 m/z.IDA number is 30, EPI scanning
Range: 100-1500 m/z；Dynamic exclusion time:20 s；Rolling CE:enabled；CES:5.
The distinguishing method between true and false of bird's nest of the present invention based on mass spectrum proteomic techniques, wherein stepIt is described
Bird's nest and its common adulterant specific peptide segment mark object in, the specific peptide segment mark object 1 and pigskin of the pigskin of pigskin
Specific peptide segment mark object 2 can also serve as the specific peptide segment mark object of pig gelatin.
The distinguishing method between true and false of bird's nest of the present invention based on mass spectrum proteomic techniques, wherein stepIt is described
Protein search in the identification of library, for bird's nest and its common adulterant pigskin, tremella, egg white and the identification of fish maw protein matter
Fasta form database derives from UniprotKB, and Taxonomy search key is specifically such as table 2:
2 bird's nest of table and its common adulterant protein identification database retrieve word list
Detailed description of the invention
Fig. 1 is that the MRM of bird's nest, pigskin, tremella, egg white and air bladder specific peptide segment mark object extracts ion figure.
Fig. 2 is the MRM verifying of tremella, pigskin, egg white and air bladder specific peptide segment mark object in bird's nest, wherein Fig. 2-1:
The MRM of two specific peptide segment mark objects of bird's nest extracts ion figure；Fig. 2-2: 1 SLWSCPYR of bird's nest specific peptide segment mark object
MS/MS figure；Fig. 2-3: 2 EMVAAFEQEAR of bird's nest specific peptide segment mark object MS/MS figure.
Fig. 3 is the MRM verifying of bird's nest, tremella, egg white and air bladder specific peptide segment mark object in pigskin, wherein Fig. 3-1:
The MRM of two specific peptide segment mark objects of pigskin extracts ion figure；Fig. 3-2: pigskin specific peptide segment mark object 1
The MS/MS of GETGPAGPAGPVGPVGAR schemes；Fig. 3-3: the MS/ of 2 TGETGASGPPGFAGEK of pigskin specific peptide segment mark object
MS figure.
The MRM of Fig. 4 bird's nest, pigskin, egg white and air bladder specific peptide segment mark object in tremella is verified, wherein Fig. 4-1:
The MRM of two specific peptide segment mark objects of tremella extracts ion figure；Fig. 4-2: tremella specific peptide segment mark object 1
The MS/MS of LITQLIEQLNAAK schemes；Fig. 4-3: 2 IPVGPATLGR of tremella specific peptide segment mark object MS/MS figure.
The MRM of Fig. 5 bird's nest, pigskin, tremella and air bladder specific peptide segment mark object in egg white is verified, wherein Fig. 5-1:
The MRM of two specific peptide segment mark objects of egg white extracts ion figure；Fig. 5-2: egg white specific peptide segment mark object 1
The MS/MS of GGLEPINFQTAADQAR schemes；Fig. 5-3: 2 VASMASEK of egg white specific peptide segment mark object MS/MS figure.
The MRM of Fig. 6 bird's nest, pigskin, tremella and egg white specific peptide segment mark object in air bladder is verified, wherein Fig. 6-1:
The MRM of two specific peptide segment mark objects of air bladder extracts ion figure；Fig. 6-2: air bladder specific peptide segment mark object 1
The MS/MS of GESGPAGPAGAAGPAGPR schemes；Fig. 6-3: 2 GLEGNAGR of air bladder specific peptide segment mark object MS/MS figure.
Note: in Fig. 1 ~ Fig. 6,: EMVAAFEQEAR；: SLWSCPYR；: TGETGASGPPGFAGEK；:
GETGPAGPAGPVGPVGAR；: IPVGPATLGR；: LITQLIEQLNAAK；: VASMASEK；:
GGLEPINFQTAADQAR；: GLEGNAGR；: GESGPAGPAGAAGPAGPR.
Specific embodiment
The present invention is further illustrated by way of embodiment, but the present invention is not limited only to following reality
Apply example.
Embodiment 1: the excavation of bird's nest and its common adulterant pigskin, tremella, egg white and air bladder specific peptide segment mark object
1. sample collect: Malaysian hole swallow, Malaysia room swallow, Indonesia hole swallow, Indonesia room swallow, Thailand hole swallow and
Thailand room swallow is entrusted by state swallow and is provided.Fried pigskin, tremella, egg, crucian air bladder, carp air bladder, black carp air bladder, blunt snout bream fish
Fish glue, grass carp air bladder and yellow croaker air bladder is dry is purchased from supermarket.The sample excavated for special peptide fragment collects 3 biological repeating samples
Product, wherein air bladder sample collected it is cheap, in the market common fish species crucian, carp, black carp, blunt snout bream, grass carp and
The air bladder of 6 kinds of fish such as yellow croaker, to guarantee the representativeness of specific peptide segment mark object.
2. sample pre-treatments
The pre-treatment of 2.1 fried pigskin samples: after fried pigskin is crushed with tissue pulverizer, about 20 grams of fried pigskin powder are taken
Be added after 200 mL pre-cooling acetone soak half an hour after being filtered to remove acetone (being repeated 3 times), liquid nitrogen grinding of volatilizing to the greatest extent after acetone at
Powder, -20 DEG C of preservations.
The pre-treatment of 2.2 bird's nests, tremella and air bladder dry-eye disease: tissue pulverizer crush after with liquid nitrogen grinding at powder, -20 DEG C
It saves.
2.3 egg white sample pre-treatments: after the egg white in egg is separated with yolk, egg white being dispensed, -20 DEG C of preservations.
3. Protein Extraction: taking bird's nest and its common adulterant pigskin, tremella, egg white and 0.2 g of air bladder in 40 mL respectively
In round bottom centrifuge tube, the aqueous solution of 10mL urea containing 7M and 2M thiocarbamide is added, in ice bath after 2 h of ultrasound assisted extraction protein, 4
12000 g are centrifuged 10 min and discard precipitating collection supernatant at DEG C, cross 4 DEG C of preservations after 0.22 μm of filter membrane.
4. the measurement (Bradford method) of protein concentration: dissolving bovine serum albumin(BSA) (BSA) with 0.15 M NaCl, match
The BSA Standard Stock solutions of 1 mg/mL are made, with phosphate buffer solution by BSA Standard Stock solutions be diluted to 1.0 mg/mL,
The concentration gradient of 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, 0.2 mg/mL, 0.1 mg/mL draw the standard of protein
Curve.It takes 30 μ L protein standard solutions and solution to be measured in 1.5 mL centrifuge tubes respectively, 1 mL developing solution (0.01% is added
Coomassie brilliant blue G250,8.5% phosphoric acid and 4.75% ethyl alcohol) be vortexed after, take 300 μ L develop the color solution in 96 hole elisa Plates,
Absorbance is surveyed under A595nm, protein content in sample is calculated according to standard curve.
5. the preparation of tryptic digestion peptide fragment: take the extracting solution of protein containing 200 μ g proteins in 1.5mL brown from
In heart pipe, 37 DEG C of water-baths after 10 μ L120 mM dithiothreitol (DTT)s (DTT) are vortexed are added and restore 1 h, 10 μ L60 are added after cooling
15 min of room temperature alkylated reaction after the iodoacetamide (IAA) of mM is vortexed.Protein solution after alkylation is transferred completely into
In 10 kDa ultra-filtration centrifuge tubes, 12000 g are centrifuged 20 min and discard the solution in collecting pipe, then 100 μ are added into super filter tube
12000 g of L50 mM ammonium bicarbonate soln centrifugation, 20 min discard the solution (repeating this operation 2 times) in collecting pipe.By ultrafiltration membrane
On solution all centrifugation go down after 100 μ L50 mM ammonium bicarbonate solns are added, in 1:50 ratio be added 1 μ g/ μ L pancreas
Protein enzyme solution (is dissolved in 50 mM acetic acid), 37 DEG C of overnight digestions.After the completion of digestion, 12000 g are centrifuged 20 min and collect filter
Liquid.12000 g after 100 μ L25 mM ammonium bicarbonate solns are vortexed are added into ultra-filtration centrifuge tube and are centrifuged 20 min collection filtrate
(being repeated once), merging filtrate.Whole filtrates are placed in traditional vacuum drying instrument, be added after dry 200 μ L ultrapure water vacuum from
The heart is dry (being repeated once), to remove volatility salt ammonium hydrogen carbonate.Peptide fragment after desalination contains 2% acetonitrile and 0.1% with 100 μ L
After the aqueous dissolution of formic acid, after 12000 g are centrifuged 5 min, take 90 μ L for mass spectral analysis.
6. NanoLC high-resolution tandem mass spectrum identifies protein: NanoLC-MS/MS high-resolution tandem mass spectrum (Eksigent
400 and TripleTOFTM 5600), Q3- IDA-EPI scanning mode.Eksigent chip chromatographic column (ChromXP C18-CL,
75 μ m 15cm, 3 μm of partial sizes, 120), mobile phase A: 2% acetonitrile, 0.1% formic acid, 98% water；Mobile phase B: 98% acetonitrile,
0.1% formic acid, 2% water；0.3 μ L/min of flow velocity, 4 μ L of sampling volume, mobile phase gradient are shown in Table 3.Loading Pump stream
2 μ L/min, 10min of speed.ESI ion source, positive ion mode, Ion spray voltage:2.4 kv；GS1:6；Curtain
Gas:30；DP:100；Q1 scanning range: 350-1250 m/z.IDA number is 30, EPI scanning range: 100-1500 m/z,
Dynamic exclusion time:20 s, Rolling CE:enabled, CES:5.
3 NanoLC mobile phase gradient program of table
5.0 parameter setting of 7.Proteinpilot:
Sample Type: Identification; Cys Alkylation: Iodoacetic acid; Digestion:
Trypsin; Search Effort: Rapid ID; ID Focus: Biological modifications。
8. the MRM of special peptide fragment is verified: using 5.0 software of Proteinpilot to the height of bird's nest and its common adulterant
Resolution mass spectra carries out searching library identification protein, and Response to selection is high, cuts without leakage, the peptide fragment without modification, length < 25 amino acid
Blast comparison is carried out on NCBI, and is verified in negative sample, the bird's nest excavated and its common adulterant pig
Skin, tremella, the specific peptide segment mark object of egg white and air bladder are as follows:
1 SLWSCPYR of specific peptide segment mark object of bird's nest
2 EMVAAFEQEAR of specific peptide segment mark object of bird's nest
1 LITQLIEQLNAAK of specific peptide segment mark object of tremella
2 IPVGPATLGR of specific peptide segment mark object of tremella
1 GETGPAGPAGPVGPVGAR of specific peptide segment mark object of pigskin
2 TGETGASGPPGFAGEK of specific peptide segment mark object of pigskin
1 GGLEPINFQTAADQAR of specific peptide segment mark object of egg white
2 VASMASEK of specific peptide segment mark object of egg white
1 GESGPAGPAGAAGPAGPR of specific peptide segment mark object of air bladder
2 GLEGNAGR of specific peptide segment mark object of air bladder
The pigskin of bird's nest and its common adulterant in MRM method, tremella, 3 pairs of specific peptide segment mark object of egg white and air bladder
MRM ion pair and its impact energy such as table 4:
4 bird's nest of table and its common adulterant specific peptide segment mark object MRM ion pair parameter
5500 operating condition of LC-Q-Trap: the XBridge peptide BEH C18 liquid-phase chromatographic column of Waters company
(4.6mm × 150mm, 3.5 μm of partial size, aperture 300), flow velocity 0.6mL/min, 40 DEG C of column temperature.Mobile phase A: containing 0.1% formic acid,
The aqueous solution of 2% acetonitrile；Mobile phase B: the acetonitrile solution containing 0.1% formic acid.Eluent gradient elution program is as shown in table 5:
5 eluent gradient elution program of table
5500 tandem mass spectrometer of AB SCIEX company Q-TRAP, ESI+ ion source, MRM scan pattern, gas curtain gas: 40；Ion source
Temperature: 600 DEG C；Gas1:60；Gas2:60；Spray voltage: 5500；DP value: 100.
Although specific embodiments of the present invention are described, those skilled in the art will appreciate that
The present invention can be variously changed and be modified under the premise of without departing from the scope or spirit of the invention.Thus, the present invention
It is intended to cover all these changes fallen in claims and its range of equivalency and modification.
Claims (5)
1. a kind of bird's nest distinguishing method between true and false based on mass spectrum proteomic techniques, with bird's nest and its common adulterant pigskin, silver
Ear, egg white and air bladder specific peptide segment mark object are target, while to adulterant pigskin common in bird's nest, tremella, egg white and fish
The method of fish glue qualitative detection, it is characterised in that: the specificity of the pigskin of bird's nest and its common adulterant, tremella, egg white and air bladder
The amino acid sequence of peptide fragment marker is as follows:
。
2. a kind of bird's nest distinguishing method between true and false based on mass spectrum proteomic techniques according to claim 1, feature exist
In 3 pairs of the specific peptide segment mark object of: the pigskin of bird's nest and its common adulterant in MRM method, tremella, egg white and air bladder
MRM ion pair and its impact energy (CE value) are as follows:
。
3. a kind of bird's nest distinguishing method between true and false based on mass spectrum proteomic techniques according to claim 1, feature exist
In the distinguishing method between true and false of the bird's nest of the present invention based on mass spectrum proteomic techniques, wherein stepThe high score
Distinguish that tandem mass spectrum (NanoLC-Q-TOF) analysis condition is as follows:
NanoLC-MS/MS high-resolution tandem mass spectrum (Eksigent 400 and TripleTOFTM 5600), Q3- IDA-EPI scanning
Mode；
Eksigent chip chromatographic column (ChromXP C18-CL, 75 μ m, 15 cm, 3 μm of partial sizes, 120), mobile phase A: 2%
Acetonitrile, 0.1% formic acid, 98% water；Mobile phase B: 98% acetonitrile, 0.1% formic acid, 2% water；0.3 μ L/min of flow velocity, sampling volume 4
μ L, mobile phase gradient are as follows:
Loading Pump flow velocity 2 μ L/min, 10min；
ESI ion source, positive ion mode, Ion spray voltage:2.4 kv；GS1:6；Curtain gas:30；DP:
100；Q1 scanning range: 350-1250 m/z；
IDA number is 30, EPI scanning range: 100-1500 m/z；Dynamic exclusion time:20 s；Rolling
CE:enabled；CES:5.
4. a kind of bird's nest distinguishing method between true and false based on mass spectrum proteomic techniques according to claim 1, feature exist
In the distinguishing method between true and false of the bird's nest of the present invention based on mass spectrum proteomic techniques, wherein stepThe bird's nest
And its in the specific peptide segment mark object of common adulterant, the specific peptide segment mark object 1 of the pigskin of pigskin and pigskin it is special
Property peptide fragment marker 2 can also serve as the specific peptide segment mark object of pig gelatin.
5. a kind of bird's nest distinguishing method between true and false based on mass spectrum proteomic techniques according to claim 1, feature exist
In the distinguishing method between true and false of the bird's nest of the present invention based on mass spectrum proteomic techniques, wherein stepThe albumen
Matter is searched in the identification of library, the Fasta lattice identified for bird's nest and its common adulterant pigskin, tremella, egg white and fish maw protein matter
For formula database source in UniprotKB, species search key is as follows:
Bird's nest Fasta form database Apodidiae
Pigskin Fasta form database Sus
Tremella Fasta form database Tremella
Egg white Fasta form database Gallus
Air bladder Fasta form database Actinopterygii.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111693625A (en) * | 2020-06-23 | 2020-09-22 | 中山市食品药品检验所 | GC-IMS-based method for identifying adulterants in cubilose and application |
| WO2022028363A1 (en) * | 2020-08-03 | 2022-02-10 | Hong Kong Baptist University | Peptide markers for authentication of edible bird's nest and related products |
| CN116297976A (en) * | 2023-03-31 | 2023-06-23 | 山东省食品药品检验研究院 | Urokinase characteristic polypeptide group and method for detecting urokinase adulteration |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| HK1147388A2 (en) * | 2011-04-08 | 2011-08-05 | Univ Hong Kong Science & Techn | A detection method for identification of the quality of edible bird's nest |
| CN102621329A (en) * | 2012-03-16 | 2012-08-01 | 深圳市计量质量检测研究院 | Bird's-nest enzyme linked immunosorbent assay kit |
| CN104360004A (en) * | 2014-12-12 | 2015-02-18 | 徐敦明 | Method for combining LC-Q-TOF ((liquid chromatography-quadrupole-time offlight mass spectrometer) with statistic analysis to identify true-false of edible bird nest |
| WO2016157250A1 (en) * | 2015-03-30 | 2016-10-06 | 国立大学法人神戸大学 | Animal species distinguishing method and device |
| CN106018328A (en) * | 2016-04-13 | 2016-10-12 | 广东出入境检验检疫局检验检疫技术中心 | Method for differentiating true and false edible bird's nests |
| CN106636345A (en) * | 2016-10-28 | 2017-05-10 | 肇庆学院 | Real-time fluorescent quantitative PCR (polymerase chain reaction) identification reagent kit and identification method for bird's nest products |
| WO2017155471A1 (en) * | 2016-03-11 | 2017-09-14 | Lim Kah Meng | Edible bird's nest extract and method of extraction |
-
2017
- 2017-12-19 CN CN201711374365.0A patent/CN109557228A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| HK1147388A2 (en) * | 2011-04-08 | 2011-08-05 | Univ Hong Kong Science & Techn | A detection method for identification of the quality of edible bird's nest |
| CN102621329A (en) * | 2012-03-16 | 2012-08-01 | 深圳市计量质量检测研究院 | Bird's-nest enzyme linked immunosorbent assay kit |
| CN104360004A (en) * | 2014-12-12 | 2015-02-18 | 徐敦明 | Method for combining LC-Q-TOF ((liquid chromatography-quadrupole-time offlight mass spectrometer) with statistic analysis to identify true-false of edible bird nest |
| WO2016157250A1 (en) * | 2015-03-30 | 2016-10-06 | 国立大学法人神戸大学 | Animal species distinguishing method and device |
| WO2017155471A1 (en) * | 2016-03-11 | 2017-09-14 | Lim Kah Meng | Edible bird's nest extract and method of extraction |
| CN106018328A (en) * | 2016-04-13 | 2016-10-12 | 广东出入境检验检疫局检验检疫技术中心 | Method for differentiating true and false edible bird's nests |
| CN106636345A (en) * | 2016-10-28 | 2017-05-10 | 肇庆学院 | Real-time fluorescent quantitative PCR (polymerase chain reaction) identification reagent kit and identification method for bird's nest products |
Non-Patent Citations (5)
| Title |
|---|
| CHUN-FAI WONG ET AL.: "Characterization of edible bird’s nest by peptide fingerprinting with principal component analysis", 《FOOD QUALITY AND SAFETY》 * |
| LILI GUO ET AL.: "Determination of edible bird’s nests by FTIR and SDS-PAGE coupled with multivariate analysis", 《FOOD CONTROL》 * |
| MERCK: "ZIC-HILIC Separation of tryptic peptides from Ovalbumin", 《SEQUANT APPLICATION NOTE 2700-46A》 * |
| 刘琳 等: "燕窝酶解产物中多肽的液相色谱质谱分析", 《中国中药杂志》 * |
| 李耿 等: "HPLC-ESI-MS联用法分析血燕与白燕水解多肽研究", 《世界科学技术-中医药现代化》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111693625A (en) * | 2020-06-23 | 2020-09-22 | 中山市食品药品检验所 | GC-IMS-based method for identifying adulterants in cubilose and application |
| WO2022028363A1 (en) * | 2020-08-03 | 2022-02-10 | Hong Kong Baptist University | Peptide markers for authentication of edible bird's nest and related products |
| CN116297976A (en) * | 2023-03-31 | 2023-06-23 | 山东省食品药品检验研究院 | Urokinase characteristic polypeptide group and method for detecting urokinase adulteration |
| CN116297976B (en) * | 2023-03-31 | 2023-09-19 | 山东省食品药品检验研究院 | Urokinase characteristic polypeptide group and method for detecting urokinase adulteration |
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