CN109293742B - Polypeptide for identifying donkey-hide gelatin and mule skin-derived component in donkey-hide gelatin product - Google Patents

Polypeptide for identifying donkey-hide gelatin and mule skin-derived component in donkey-hide gelatin product Download PDF

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CN109293742B
CN109293742B CN201811245616.XA CN201811245616A CN109293742B CN 109293742 B CN109293742 B CN 109293742B CN 201811245616 A CN201811245616 A CN 201811245616A CN 109293742 B CN109293742 B CN 109293742B
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CN109293742A (en
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张鸿伟
张晓梅
赵飒
王培锋
周祥山
王境堂
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Qingdao Customs Technology Center
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Abstract

The invention relates to a group of polypeptides for identifying mule skin-derived components in donkey-hide gelatin and donkey-hide gelatin-containing products, and a method for identifying mule skin-derived components in donkey-hide gelatin and donkey-hide gelatin-containing products by using the polypeptides through mass spectrometry, wherein the polypeptides in the polypeptide group have a specific sequence structure and a specific m/z value (including a specific parent ion and a pair of daughter ions), the sequences of the polypeptides are shown as SEQ ID NO. 1-36, the peptide sections SEQ ID NO.1-30 are common to horse skin and mule skin-derived gelatin, and the donkey skin gelatin, the mule skin-derived component and the mule skin-derived component are not present in boiled gelatin; the peptide segments SEQ ID NO.31-36 and m/z thereof are common to mule hide-derived glue and donkey-hide gelatin, and no mule hide-derived glue, pig hide-derived glue and horse hide-derived glue are produced.

Description

Polypeptide for identifying donkey-hide gelatin and mule skin-derived component in donkey-hide gelatin product
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a group of polypeptides for identifying donkey-hide gelatin and mule skin-derived components in a donkey-hide gelatin product.
Background
Donkey-hide gelatin is a solid gelatin prepared by decocting and concentrating the skin of donkey of an equine family, is originally produced in Dong-A county of Shandong province, and has been used for nearly three thousand years. The donkey-hide gelatin is a traditional nourishing top-grade and blood-enriching holy medicine, is sweet and mild in taste, enters lung, liver and kidney channels, has the effects of enriching blood and stopping bleeding, nourishing yin and moistening dryness and the like, is used as both medicine and food, can enrich blood and nourish blood, whiten and beautify skin, resist aging, resist fatigue and improve immunity after being taken for a long time, and is widely applicable to people. Li Shizhen Bin (compendium of materia Medica) Bing: "E jiao" Ben Jing "the best quality. The Chinese herbal medicine has the following characteristics: 'Shandong donkey-hide gelatin' is known for its name. Donkey-hide gelatin is the most specific "genuine" herb among the herbs, and genuine donkey-hide gelatin must absorb Dong's water and be refined by the strange skill of the inheritor. The genuine donkey-hide gelatin has smooth surface, no pores, hard and crisp texture, bright and fine section, and fragments are in brown semitransparent shapes to light, and Li Shizhenzan has yellow color like amber color and black color like paint.
Pseudo-colla Corii Asini events were exposed as early as 1996. After many years, donkey-hide gelatin counterfeiting is not only not inhibited but also increasingly vigorous. These illegal enterprises use inferior materials such as cowhide, horse hide, pigskin, mule hide, etc. as leftovers to make fake donkey-hide gelatin by imitating donkey hide, and sell the fake donkey-hide gelatin to market after being supplemented with the inferior materials.
Part of the reasons that donkey-hide gelatin is frequently forbidden are lack of technical support for identification, and the source of the animal skin is difficult to identify because the characteristics of the animal skin are destroyed after the animal skin is decocted and dissolved. At present, no method for effectively identifying the donkey-hide gelatin component exists in China, and the donkey-hide gelatin component can be controlled only by a production source, so that a plurality of illegal enterprises are hollowed out.
A method for efficiently and accurately identifying the authenticity of the donkey-hide gelatin and products containing the donkey-hide gelatin is urgently needed. With the development of metabonomics technology, it became possible to directly identify animal-derived components using peptide biology.
Disclosure of Invention
The invention researches the polypeptide in donkey-hide gelatin and mule skin gelatin, verifies the polypeptide in cow skin gelatin, pig skin gelatin and horse skin gelatin, establishes the technology for identifying mule-derived components in donkey-hide gelatin and products containing donkey-hide gelatin from the polypeptide level, and fills the blank of the identification of donkey-hide gelatin and products containing donkey-hide gelatin at home and abroad.
The invention firstly relates to a group of polypeptides for detecting alone or in combination the mule skin source components in donkey-hide gelatin and donkey-hide gelatin-containing products, wherein the donkey-hide gelatin-containing products comprise, but are not limited to, donkey-hide gelatin cakes, donkey-hide gelatin pastes, donkey-hide gelatin slurries, donkey-hide gelatin oral liquids, donkey-hide gelatin sugars and donkey-hide gelatin blood-enriching particles, the polypeptide sequence is that the peptide sections 1-30 are mule skin source and horse skin source shared polypeptides, and the peptide sections 31-36 are mule skin source and donkey-hide gelatin shared polypeptides:
peptide fragment 1: SEQ ID NO. 1: LQTEAGEYSR
Peptide fragment 2: SEQ ID NO. 2: LVNDLTGQR, respectively;
peptide fragment 3: SEQ ID NO. 3: GSLGGGFSSGGFSGGSFSR, respectively;
peptide fragment 4: SEQ ID NO. 4: ELNMDNILVEIK, respectively;
peptide fragment 5: SEQ ID No. 5: FFDSFGDLSNPGAVMGN, respectively;
peptide fragment 6: SEQ ID NO. 6: PGAVMGNPK, respectively;
peptide fragment 7: SEQ ID NO. 7: DYAQVGR;
peptide fragment 8: SEQ ID NO. 8: SSSGQSSGFGR, respectively;
peptide fragment 9: SEQ ID NO. 9: MELETAGR;
peptide fragment 10: SEQ ID NO. 10: LPTGLPVSLLTLYLDNNK, respectively;
peptide fragment 11: SEQ ID NO. 11: YSSGSGAYSSGGR, respectively;
peptide fragment 12: SEQ ID NO. 12: ISGAGTGFGSR, respectively;
peptide fragment 13: SEQ ID NO. 13: GPSGEPGKPGDKGHAGLAGAR, respectively;
peptide fragment 14: SEQ ID No. 14: PVPHGTGSVPESPR, respectively;
peptide fragment 15: SEQ ID NO. 15: AEAESWYQSK, respectively;
peptide fragment 16: SEQ ID No. 16: VPSILDWVQK, respectively;
peptide fragment 17: SEQ ID NO. 17: VGYVSGWGR, respectively;
peptide fragment 18: SEQ ID NO. 18: DYAQVGR;
peptide fragment 19: SEQ ID NO. 19: NPVDQVQR;
peptide fragment 20: SEQ ID No. 20: QLEDELVSLQK, respectively;
peptide fragment 21: SEQ ID NO. 21: PGAVMGNPK, respectively;
peptide fragment 22: SEQ ID NO. 22: GSLGGGFSSGGFSGGSFSR, respectively;
peptide fragment 23: SEQ ID NO. 23: SLGGGFSSGGFSGGSFSR, respectively;
peptide fragment 24: SEQ ID No. 24: GETGPAGPAGPVGPVGAR, respectively;
peptide fragment 25: SEQ ID No. 25: GEPGPPGEAGAAGPAGNPGADGQPGAK, respectively;
peptide fragment 26: SEQ ID NO. 26: EGPVGLPGIDGRSGPIGPAGPR, respectively;
peptide fragment 27: SEQ ID NO. 27: GEPGPPGPAGFAGPPGADGQPGAK, respectively;
peptide fragment 28: SEQ ID NO. 28: ELNMDNILAEIK, respectively;
peptide fragment 29: SEQ ID NO. 29: VFVDLIR;
peptide fragment 30: SEQ ID No. 30: VVFHPDYQEVDIGLIK, respectively;
peptide fragment 31: SEQ ID NO. 31: GEPGGAGPVGPPGER, respectively;
peptide fragment 32: SEQ ID NO. 32: GSGGAAGVPGER, respectively;
peptide fragment 33: SEQ ID NO. 33: TGPAGAAGAR, respectively;
peptide fragment 34: SEQ ID No. 34: GLLESEDGKLPR R, respectively;
peptide fragment 35: SEQ ID NO. 35: TGPAGAAGAR, respectively;
peptide fragment 36: SEQ ID NO. 36: GLLESEDGK are provided.
The m/z is respectively as follows:
peptide fragment 1: 577.3;
peptide fragment 2: 508.3;
peptide fragment 3: 854.4, respectively;
peptide fragment 4: 715.9, respectively;
peptide fragment 5: 887.9, respectively;
peptide fragment 6: 435.7, respectively;
peptide fragment 7: 404.7;
peptide fragment 8: 528.7, respectively;
peptide fragment 9: 453.7, respectively;
peptide fragment 10: 986.5, respectively;
peptide fragment 11: 618.2, respectively;
peptide fragment 12: 505.2;
peptide fragment 13: 644.6, respectively;
peptide fragment 14: 472.9;
peptide fragment 15: 600.1;
peptide fragment 16: 593.2, respectively;
peptide fragment 17: 491.0, respectively;
peptide fragment 18: 404.9, respectively;
peptide fragment 19: 478.5, respectively;
peptide fragment 20: 651.7, respectively;
peptide fragment 21: 436.0;
peptide fragment 22: 854.9, respectively;
peptide fragment 23: 855.3, respectively;
peptide fragment 24: 523.5;
peptide fragment 25: 1142.5, respectively;
peptide fragment 26: 691.3, respectively;
peptide fragment 27: 701.3;
peptide fragment 28: 701.8, respectively;
peptide fragment 29: 431.2, respectively;
peptide fragment 30: 625.0, respectively;
peptide fragment 31: 683.3, respectively;
peptide fragment 32: 515.7, respectively;
peptide fragment 33: 414.7, respectively;
peptide fragment 34: 438.5;
peptide fragment 35: 414.9, respectively;
peptide fragment 36: 474.2.
the parent ion and partial daughter ion corresponding to 36 polypeptides are shown in the following table 1,
TABLE 1 mule skin-derived polypeptide sequences and their corresponding parent ions, partial daughter ions
Figure GDA0003546161410000031
Figure GDA0003546161410000041
Figure GDA0003546161410000051
The pretreatment method comprises the following steps:
(1) performing mass spectrum pretreatment on a sample to be detected to obtain polypeptide filtrate to be detected:
(2) detecting polypeptide components of a sample to be detected by mass spectrometry, and analyzing animal skin-derived components in the sample.
The mass spectrum pretreatment steps are as follows:
(1) homogenizing the sample to be tested into powder, weighing 0.1-0.5 g colla Corii Asini sample or 1.0-2.0g colla Corii Asini product, and adding 1% NH4HCO3Carrying out ultrasonic treatment on the solution for 10-30min to completely dissolve the sample, and fixing the volume to 50 mL;
(2) filtering the solution through a 0.22 mu m filter membrane;
(3) adding 10-20 mu L of Trypsin enzyme solution (1 mu g/mu L of Trypsin enzyme solution) into 100 mu L-200 mu L of the filtrate, carrying out enzymolysis at 37 ℃ for 16-18 hours, and waiting for detection on a computer.
The invention also relates to two mass spectrum methods for detecting the donkey-hide gelatin and the donkey-hide gelatin-containing products, wherein the mass spectrum method comprises the following steps,
using AB SCIEX
Figure GDA0003546161410000052
5600 the mass spectrometric detection is carried out,
mobile phase A: 0.05-0.2% formic acid-acetonitrile solution, mobile phase B: 0.05 to 0.2% formic acid-water,
flow rate: 0.1 to 0.5mL/min,
TOF scan range: 100-3000Da of the total weight of the material,
positive ion reaction mode, GS 1: 35, GS 2: 45, Curtain Gas: 35, ISVF: 5500, TEM: 500, DP: 100, CE: 10.
using AB SCIEX triple quadrupole mass spectrometry for detection,
mobile phase A: 0.05-0.2% formic acid-acetonitrile, mobile phase B: 0.05 to 0.2% formic acid-water,
flow rate: 0.1 to 0.5mL/min,
electrospray ion source, positive ion reaction mode, detection mode: MRM, spray voltage: 5500V, ion transfer tube temperature: 475 ℃; sheath gas pressure: 40; auxiliary gas pressure: 6.
the mule skin-derived component in the analysis sample is characterized in that the mule skin-derived component in the tissue sample can be judged to exist if the mass spectrum detection of the specific polypeptides of SEQ ID NO.1-30 exists and the mule skin-derived component does not exist when the mass spectrum result of the sample to be detected is compared with the mass spectrum spectrograms of the specific polypeptides of SEQ ID NO. 1-36 and the mass spectrum detection spectrograms of the specific polypeptides of SEQ ID NO.31-36 appear; if the mass spectrum detection of the specific polypeptide of SEQ ID NO.1-30 does not exist, judging that donkey skin derived components exist in the tissue sample; if the mass spectrometric detection of each specific polypeptide of SEQ ID No.31-36 does not exist, the tissue sample does not contain mule skin-derived and donkey skin-derived components.
Drawings
FIG. 1 shows a chromatogram characteristic diagram for detecting polypeptides shown in SEQ ID NO. 1-36 in donkey-hide gelatin
FIG. 2 is a chromatogram characteristic diagram for detecting polypeptides shown in SEQ ID NO. 1-36 in corium elephatis
FIG. 3 is a chromatogram characteristic diagram for detecting polypeptides shown in SEQ ID NO. 1-36 in oxhide glue
FIG. 4 is a chromatogram characteristic diagram for detecting the polypeptides shown in SEQ ID NO. 1-36 in pigskin glue
FIG. 5 is a chromatogram characteristic diagram for detecting polypeptides shown in SEQ ID NO. 1-36 in mule skin glue
FIG. 6 shows the mass spectrum of the representative characteristic peptide of mule-hide gelatin and donkey-hide gelatin in donkey-hide gelatin
FIG. 7 mass spectrogram of representative characteristic peptide of mule-hide gelatin and donkey-hide gelatin in mule-hide gelatin
FIG. 8 mass spectrum of representative characteristic peptide of mule hide gelatin and donkey hide gelatin in cow hide gelatin
FIG. 9 mass spectrum of representative characteristic peptide of mule hide gelatin and donkey hide gelatin in pigskin gelatin
FIG. 10 shows the mass spectra of representative characteristic peptide of mule-hide gelatin and donkey-hide gelatin in horse-hide gelatin
FIG. 11 shows the mass spectrum of representative characteristic peptide of mule-hide gelatin and horse-hide gelatin in donkey-hide gelatin
FIG. 12 is a mass spectrum of representative characteristic peptide of mule skin glue and mao skin glue in mule skin glue
FIG. 13 mass spectrum of representative characteristic peptide of mule hide gelatin and horse hide gelatin in cow hide gelatin
FIG. 14 mass spectrum of representative characteristic peptide of mule hide gelatin and horse hide gelatin in pigskin gelatin
FIG. 15 shows the mass spectra of representative characteristic peptide of mule hide glue and Ma hide glue in Ma hide glue
FIG. 16, mass spectrum of characteristic peptide of mule hide gelatin and donkey hide gelatin in sample 1
FIG. 17, the peptide mass spectrum of mule hide glue and massa hide glue in sample 1
FIG. 18, mass spectrum of horse hide glue characteristic peptide in sample 1
FIG. 19 is a mass spectrum of characteristic peptides of mule hide gelatin and donkey hide gelatin in control
FIG. 20 is a chart showing the characteristic peptide mass spectra of mule hide glue and massa hide glue in the control
Detailed Description
Example 1 screening of equine Peel rubber-specific Polypeptides
1. Through mass spectrometry analysis of pure product mule skin glue samples and donkey skin donkey hide glue samples, the protein Pilot software is utilized to search, compare and analyze mass spectrometry results, no mule skin glue specific characteristic polypeptide is found, but the most preferable common specific characteristic polypeptide sequence of mule skin glue and donkey skin donkey hide glue is determined, namely SEQ ID NO. 1-30.
2. Because mules are the offspring of horses and donkeys and have affinity, the same method is used for analyzing the polypeptide of the horse leather and the mule leather, but the special characteristic polypeptide of the mule leather is not found, but the special characteristic polypeptide sequence SEQ ID NO.31-36 shared by the most preferred mule leather and the mule leather is determined.
The specific mass spectrometry method is as follows:
firstly, performing mass spectrometry on a selected pure gelatin sample, wherein the steps comprise:
(I) sample pretreatment:
(1) weighing 0.1-0.5 g of pure sample homogenized into powder, and adding 1% NH4HCO3Carrying out ultrasonic treatment on the solution for 10-30min to completely dissolve the sample, and fixing the volume to 50 mL;
(2) filtering the solution through a 0.22 mu m filter membrane;
(3) adding 10-20 mu L of Trypsin enzyme solution (1 mu g/mu L of Trypsin enzyme solution) into 100 mu L-200 mu L of the filtrate, carrying out enzymolysis at 37 ℃ for 16-18 hours, and waiting for detection on a computer.
(II) performing on-machine detection,
(1) using AB SCIEX
Figure GDA0003546161410000071
5600 the method of mass spectrometric detection is as follows,
mobile phase A: 0.05-0.2% formic acid-acetonitrile solution, mobile phase B: 0.05 to 0.2% formic acid-water,
flow rate: 0.1 to 0.5mL/min,
TOF scan range: 100-3000Da of the total weight of the material,
positive ion reaction mode, GS 1: 35, GS 2: 45, Curtain Gas: 35, ISVF: 5500, TEM: 500, DP: 100, CE: 10.
(2) the detection method by using AB SCIEX triple quadrupole mass spectrometry is as follows,
mobile phase A: 0.05-0.2% formic acid-acetonitrile, mobile phase B: 0.05 to 0.2% formic acid-water,
flow rate: 0.1 to 0.5mL/min,
electrospray ion source, positive ion reaction mode, detection mode: MRM, spray voltage: 5500V, ion transfer tube temperature: 475 ℃; sheath gas pressure: 40; auxiliary gas pressure: 6.
secondly, according to the mass spectrum result of the pure gelatin sample, the protein Pilot software is utilized to carry out retrieval comparison analysis on the mass spectrum result,
(1) screening to obtain a common special characteristic polypeptide group which really exists in mule hide glue and donkey-hide glue, wherein the sequence information of each polypeptide in the group is as shown in SEQ ID NO. 1-30;
(2) screening to obtain a common special characteristic polypeptide group which really exists in mule hide glue and horse hide glue, wherein the sequence information of each polypeptide in the group is as follows SEQ ID NO. 31-36:
peptide fragment 1: SEQ ID NO. 1: LQTEAGEYSR
Peptide fragment 2: SEQ ID NO. 2: LVNDLTGQR, respectively;
peptide fragment 3: SEQ ID NO. 3: GSLGGGFSSGGFSGGSFSR, respectively;
peptide fragment 4: SEQ ID NO. 4: ELNMDNILVEIK, respectively;
peptide fragment 5: SEQ ID No. 5: FFDSFGDLSNPGAVMGN, respectively;
peptide fragment 6: SEQ ID NO. 6: PGAVMGNPK, respectively;
peptide fragment 7: SEQ ID NO. 7: DYAQVGR;
peptide fragment 8: SEQ ID NO. 8: SSSGQSSGFGR, respectively;
peptide fragment 9: SEQ ID NO. 9: MELETAGR;
peptide fragment 10: SEQ ID NO. 10: LPTGLPVSLLTLYLDNNK, respectively;
peptide fragment 11: SEQ ID NO. 11: YSSGSGAYSSGGR, respectively;
peptide fragment 12: SEQ ID NO. 12: ISGAGTGFGSR, respectively;
peptide fragment 13: SEQ ID NO. 13: GPSGEPGKPGDKGHAGLAGAR, respectively;
peptide fragment 14: SEQ ID No. 14: PVPHGTGSVPESPR, respectively;
peptide fragment 15: SEQ ID NO. 15: AEAESWYQSK, respectively;
peptide fragment 16: SEQ ID No. 16: VPSILDWVQK, respectively;
peptide fragment 17: SEQ ID NO. 17: VGYVSGWGR, respectively;
peptide fragment 18: SEQ ID NO. 18: DYAQVGR;
peptide fragment 19: SEQ ID NO. 19: NPVDQVQR;
peptide fragment 20: SEQ ID No. 20: QLEDELVSLQK, respectively;
peptide fragment 21: SEQ ID NO. 21: PGAVMGNPK, respectively;
peptide fragment 22: SEQ ID NO. 22: GSLGGGFSSGGFSGGSFSR, respectively;
peptide fragment 23: SEQ ID NO. 23: SLGGGFSSGGFSGGSFSR, respectively;
peptide fragment 24: SEQ ID No. 24: GETGPAGPAGPVGPVGAR, respectively;
peptide fragment 25: SEQ ID No. 25: GEPGPPGEAGAAGPAGNPGADGQPGAK, respectively;
peptide fragment 26: SEQ ID NO. 26: EGPVGLPGIDGRSGPIGPAGPR, respectively;
peptide fragment 27: SEQ ID NO. 27: GEPGPPGPAGFAGPPGADGQPGAK, respectively;
peptide fragment 28: SEQ ID NO. 28: ELNMDNILAEIK, respectively;
peptide fragment 29: SEQ ID NO. 29: VFVDLIR;
peptide fragment 30: SEQ ID No. 30: VVFHPDYQEVDIGLIK, respectively;
peptide fragment 31: SEQ ID NO. 31: GEPGGAGPVGPPGER, respectively;
peptide fragment 32: SEQ ID NO. 32: GSGGAAGVPGER, respectively;
peptide fragment 33: SEQ ID NO. 33: TGPAGAAGAR, respectively;
peptide fragment 34: SEQ ID No. 34: GLLESEDGKLPR R, respectively;
peptide fragment 35: SEQ ID NO. 35: TGPAGAAGAR, respectively;
peptide fragment 36: SEQ ID NO. 36: GLLESEDGK are provided.
The values of m/z for the 36 polypeptides are as follows:
peptide fragment 1: 577.3;
peptide fragment 2: 508.3;
peptide fragment 3: 854.4, respectively;
peptide fragment 4: 715.9, respectively;
peptide fragment 5: 887.9, respectively;
peptide fragment 6: 435.7, respectively;
peptide fragment 7: 404.7;
peptide fragment 8: 528.7, respectively;
peptide fragment 9: 453.7, respectively;
peptide fragment 10: 986.5, respectively;
peptide fragment 11: 618.2, respectively;
peptide fragment 12: 505.2;
peptide fragment 13: 644.6, respectively;
peptide fragment 14: 472.9;
peptide fragment 15: 600.1;
peptide fragment 16: 593.2, respectively;
peptide fragment 17: 491.0, respectively;
peptide fragment 18: 404.9, respectively;
peptide fragment 19: 478.5, respectively;
peptide fragment 20: 651.7, respectively;
peptide fragment 21: 436.0;
peptide fragment 22: 854.9, respectively;
peptide fragment 23: 855.3, respectively;
peptide fragment 24: 523.5;
peptide fragment 25: 1142.5, respectively;
peptide fragment 26: 691.3, respectively;
peptide fragment 27: 701.3;
peptide fragment 28: 701.8, respectively;
peptide fragment 29: 431.2, respectively;
peptide fragment 30: 625.0, respectively;
peptide fragment 31: 683.3, respectively;
peptide fragment 32: 515.7, respectively;
peptide fragment 33: 414.7, respectively;
peptide fragment 34: 438.5;
peptide fragment 35: 414.9, respectively;
peptide fragment 36: 474.2.
finally, donkey-hide gelatin samples, oxhide gelatin, pigskin gelatin, horse hide gelatin and mule hide gelatin samples are processed and detected by the same method, mass spectrum detection results are matched, and the results show that each polypeptide sample shown in SEQ ID No.1-30 does not exist in the oxhide gelatin and the pigskin gelatin; each of the polypeptide samples shown in SEQ ID nos. 31 to 36 does not exist in bovine hide gelatin or porcine hide gelatin.
FIGS. 1 to 5 are chromatogram characteristic diagrams of the polypeptide SEQ ID NO.1 to 36 detected in donkey-hide gelatin, horse hide gelatin, cow hide gelatin, pig hide gelatin and mule hide gelatin samples, and it can be seen that chromatogram peaks of SEQ ID NO.1 to 36 are not detected in cow hide gelatin and pig hide gelatin samples.
Fig. 6-10 show mass spectra of one of the representative characteristic peptide segments (SEQ ID No.25) shared by mule hide glue and donkey-hide glue in samples of mule hide glue, horse hide glue, cow hide glue, pig hide glue and mule hide glue, respectively, showing that the representative characteristic peptide segment shared by mule hide glue and donkey-hide glue is obviously present in the spectra of mule hide glue and donkey-hide glue, but is absent in the spectra of cow hide glue, pig hide glue and horse hide glue.
Fig. 11-15 show mass spectra of one of representative characteristic peptide segments (SEQ ID No.34) shared by mule hide glue and horse hide glue in samples of mule hide glue, horse hide glue, cow hide glue, pig hide glue and mule hide glue, respectively, showing that the representative characteristic peptide segment shared by mule hide glue and horse hide glue is obviously present in the mule hide glue and horse hide glue spectra, but is absent in the samples of mule hide glue, pig hide glue and donkey hide glue.
Example 2, no specific donkey-hide gelatin sample was tested to determine whether it was a fake product of horse hide gelatin
And carrying out mass spectrum analysis on the examined donkey-hide gelatin, the donkey-hide gelatin product sample and the commercially available donkey-hide gelatin product to determine whether the donkey-hide gelatin is a pure donkey-hide gelatin.
The method for treating and detecting the sample to be tested was the same as that in example 1
Step (I) sample pretreatment step:
(1) homogenizing the sample to be tested into powder, weighing 0.1g colla Corii Asini sample or 2.0g colla Corii Asini product, adding 1% NH4HCO3Carrying out ultrasonic treatment on the solution for 10-30min to completely dissolve the sample, and fixing the volume to 50 mL;
(2) filtering the solution through a 0.22 mu m filter membrane;
(3) adding 10 μ L of Trypsin enzyme solution (1 μ g/μ L of Trypsin enzyme solution) into 100 μ L of the above filtrate, performing enzymolysis at 37 deg.C for 16-18 hr, and performing detection on the machine.
In the step (II), the machine is used for detection,
using AB SCIEX
Figure GDA0003546161410000091
5600 the mass spectrometric detection is carried out,
mobile phase A: 0.1% formic acid-acetonitrile, mobile phase B: 0.1 percent of formic acid-water,
flow rate: the concentration of the active carbon is 0.25mL/min,
TOF scan range: 350-1500Da,
positive ion reaction mode, GS 1: 35, GS 2: 45, Curtain Gas: 35, ISVF: 5500, TEM: 500, DP: 100, CE: 10.
using AB SCIEX triple quadrupole mass spectrometry for detection,
mobile phase A: 0.1% formic acid-acetonitrile, mobile phase B: 0.1 percent of formic acid-water,
flow rate: 0.3mL/min of the water-soluble polymer,
electrospray ion source, positive ion reaction mode, detection mode: MRM, spray voltage: 5500V, ion transfer tube temperature: 475 ℃; sheath gas pressure: 40; auxiliary gas pressure: 6.
comparing the detection results with the mass spectrum of each specific polypeptide in example 1,
(1) when the mass spectrum detection spectrogram of each specific polypeptide of SEQ ID NO.31-36 described in example 1 appears, if the mass spectrum detection of the specific polypeptides of SEQ ID NO.1-30 exists and the specific polypeptide of the equine derived component is detected to be absent independently, the existence of the tissue sample can be judgedMule skin derived component(ii) a If the mass spectrum detection of the specific polypeptide of SEQ ID NO.1-30 exists, the existence of the tissue sample can be judgedDonkey hide derived component；
(2) And if the mass spectrum detection of each specific polypeptide of SEQ ID NO.31-36 does not exist, the tissue sample does not have mule skin-derived components and horse skin-derived components.
Sample 1 detected only mule skin-derived components:
selecting the two common characteristic polypeptides (SEQ ID No.12 and SEQ ID No.25) of the mule hide glue and the donkey-hide gelatin, the two common characteristic polypeptides (SEQ ID No.31 and SEQ ID No.34) of the mule hide glue and the donkey-hide gelatin and the known two characteristic polypeptides of the mule hide glue as references, detecting a chromatographic peak (see fig. 16) of the two common characteristic polypeptides of the mule hide glue and the donkey-hide gelatin in the sample 1 under the same detection condition, detecting a chromatographic peak (see fig. 17) of the two common characteristic polypeptides of the mule hide glue and the donkey-hide gelatin, and not detecting a chromatographic peak (see fig. 18) of the specific polypeptides of the donkey-hide gelatin; as can be seen, sample 1 contained only mule skin source components.
The reference substance only detects donkey skin-derived ingredients, and no mule skin-derived ingredients are detected:
the characteristic polypeptides (SEQ ID No.4 and SEQ ID No.25) shared by the two mule hide glues and the donkey-hide glue in the example 1 and the characteristic polypeptides (SEQ ID No.34 and SEQ ID No.36) shared by the two mule hide glues and the donkey-hide glue are selected as references, and under the same detection condition, the chromatographic peak of the characteristic polypeptides (shown in figure 19) shared by the mule hide glue and the donkey-hide glue is detected in the mahonia-jongji product produced by the reference product of the Dong donkey-hide glue, and the chromatographic peak of the characteristic polypeptides (shown in figure 20) shared by the mule hide glue and the donkey-hide glue is not detected.
Finally, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention, and should not be used to limit the protection scope of the present invention.
SEQUENCE LISTING
<110> Shandong entry-exit inspection and quarantine technical center
<120> a polypeptide for identifying donkey-hide gelatin and mule skin-derived components in donkey-hide gelatin products
<160> 36
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> PRT
<213> Artificial sequence
<400> 1
Leu Gln Thr Glu Ala Gly Glu Tyr Ser Arg
1 5 10
<210> 2
<211> 9
<212> PRT
<213> Artificial sequence
<400> 2
Leu Val Asn Asp Leu Thr Gly Gln Arg
1 5
<210> 3
<211> 19
<212> PRT
<213> Artificial sequence
<400> 3
Gly Ser Leu Gly Gly Gly Phe Ser Ser Gly Gly Phe Ser Gly Gly Ser
1 5 10 15
Phe Ser Arg
<210> 4
<211> 12
<212> PRT
<213> Artificial sequence
<400> 4
Glu Leu Asn Met Asp Asn Ile Leu Val Glu Ile Lys
1 5 10
<210> 5
<211> 17
<212> PRT
<213> Artificial sequence
<400> 5
Phe Phe Asp Ser Phe Gly Asp Leu Ser Asn Pro Gly Ala Val Met Gly
1 5 10 15
Asn
<210> 6
<211> 9
<212> PRT
<213> Artificial sequence
<400> 6
Pro Gly Ala Val Met Gly Asn Pro Lys
1 5
<210> 7
<211> 7
<212> PRT
<213> Artificial sequence
<400> 7
Asp Tyr Ala Gln Val Gly Arg
1 5
<210> 8
<211> 11
<212> PRT
<213> Artificial sequence
<400> 8
Ser Ser Ser Gly Gln Ser Ser Gly Phe Gly Arg
1 5 10
<210> 9
<211> 8
<212> PRT
<213> Artificial sequence
<400> 9
Met Glu Leu Glu Thr Ala Gly Arg
1 5
<210> 10
<211> 18
<212> PRT
<213> Artificial sequence
<400> 10
Leu Pro Thr Gly Leu Pro Val Ser Leu Leu Thr Leu Tyr Leu Asp Asn
1 5 10 15
Asn Lys
<210> 11
<211> 13
<212> PRT
<213> Artificial sequence
<400> 11
Tyr Ser Ser Gly Ser Gly Ala Tyr Ser Ser Gly Gly Arg
1 5 10
<210> 12
<211> 11
<212> PRT
<213> Artificial sequence
<400> 12
Ile Ser Gly Ala Gly Thr Gly Phe Gly Ser Arg
1 5 10
<210> 13
<211> 21
<212> PRT
<213> Artificial sequence
<400> 13
Gly Pro Ser Gly Glu Pro Gly Lys Pro Gly Asp Lys Gly His Ala Gly
1 5 10 15
Leu Ala Gly Ala Arg
20
<210> 14
<211> 14
<212> PRT
<213> Artificial sequence
<400> 14
Pro Val Pro His Gly Thr Gly Ser Val Pro Glu Ser Pro Arg
1 5 10
<210> 15
<211> 10
<212> PRT
<213> Artificial sequence
<400> 15
Ala Glu Ala Glu Ser Trp Tyr Gln Ser Lys
1 5 10
<210> 16
<211> 10
<212> PRT
<213> Artificial sequence
<400> 16
Val Pro Ser Ile Leu Asp Trp Val Gln Lys
1 5 10
<210> 17
<211> 9
<212> PRT
<213> Artificial sequence
<400> 17
Val Gly Tyr Val Ser Gly Trp Gly Arg
1 5
<210> 18
<211> 7
<212> PRT
<213> Artificial sequence
<400> 18
Asp Tyr Ala Gln Val Gly Arg
1 5
<210> 19
<211> 8
<212> PRT
<213> Artificial sequence
<400> 19
Asn Pro Val Asp Gln Val Gln Arg
1 5
<210> 20
<211> 11
<212> PRT
<213> Artificial sequence
<400> 20
Gln Leu Glu Asp Glu Leu Val Ser Leu Gln Lys
1 5 10
<210> 21
<211> 9
<212> PRT
<213> Artificial sequence
<400> 21
Pro Gly Ala Val Met Gly Asn Pro Lys
1 5
<210> 22
<211> 19
<212> PRT
<213> Artificial sequence
<400> 22
Gly Ser Leu Gly Gly Gly Phe Ser Ser Gly Gly Phe Ser Gly Gly Ser
1 5 10 15
Phe Ser Arg
<210> 23
<211> 18
<212> PRT
<213> Artificial sequence
<400> 23
Ser Leu Gly Gly Gly Phe Ser Ser Gly Gly Phe Ser Gly Gly Ser Phe
1 5 10 15
Ser Arg
<210> 24
<211> 18
<212> PRT
<213> Artificial sequence
<400> 24
Gly Glu Thr Gly Pro Ala Gly Pro Ala Gly Pro Val Gly Pro Val Gly
1 5 10 15
Ala Arg
<210> 25
<211> 27
<212> PRT
<213> Artificial sequence
<400> 25
Gly Glu Pro Gly Pro Pro Gly Glu Ala Gly Ala Ala Gly Pro Ala Gly
1 5 10 15
Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys
20 25
<210> 26
<211> 22
<212> PRT
<213> Artificial sequence
<400> 26
Glu Gly Pro Val Gly Leu Pro Gly Ile Asp Gly Arg Ser Gly Pro Ile
1 5 10 15
Gly Pro Ala Gly Pro Arg
20
<210> 27
<211> 24
<212> PRT
<213> Artificial sequence
<400> 27
Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro Pro Gly
1 5 10 15
Ala Asp Gly Gln Pro Gly Ala Lys
20
<210> 28
<211> 12
<212> PRT
<213> Artificial sequence
<400> 28
Glu Leu Asn Met Asp Asn Ile Leu Ala Glu Ile Lys
1 5 10
<210> 29
<211> 7
<212> PRT
<213> Artificial sequence
<400> 29
Val Phe Val Asp Leu Ile Arg
1 5
<210> 30
<211> 16
<212> PRT
<213> Artificial sequence
<400> 30
Val Val Phe His Pro Asp Tyr Gln Glu Val Asp Ile Gly Leu Ile Lys
1 5 10 15
<210> 31
<211> 15
<212> PRT
<213> Artificial sequence
<400> 31
Gly Glu Pro Gly Gly Ala Gly Pro Val Gly Pro Pro Gly Glu Arg
1 5 10 15
<210> 32
<211> 12
<212> PRT
<213> Artificial sequence
<400> 32
Gly Ser Gly Gly Ala Ala Gly Val Pro Gly Glu Arg
1 5 10
<210> 33
<211> 10
<212> PRT
<213> Artificial sequence
<400> 33
Thr Gly Pro Ala Gly Ala Ala Gly Ala Arg
1 5 10
<210> 34
<211> 13
<212> PRT
<213> Artificial sequence
<400> 34
Gly Leu Leu Glu Ser Glu Asp Gly Lys Leu Pro Arg Arg
1 5 10
<210> 35
<211> 10
<212> PRT
<213> Artificial sequence
<400> 35
Thr Gly Pro Ala Gly Ala Ala Gly Ala Arg
1 5 10
<210> 36
<211> 9
<212> PRT
<213> Artificial sequence
<400> 36
Gly Leu Leu Glu Ser Glu Asp Gly Lys
1 5

Claims (2)

1. The application of a group of polypeptide groups in the preparation of a detection product for detecting mule skin-derived components in donkey-hide gelatin products,
the colla Corii Asini-containing product comprises colla Corii Asini cake, colla Corii Asini paste, colla Corii Asini slurry, colla Corii Asini oral liquid, colla Corii Asini sugar, and colla Corii Asini blood tonifying granule;
the polypeptide group is as follows:
(1) peptide sections SEQ ID NO.4, 12 and 25 are mule skin source and donkey skin source shared polypeptide,
peptide fragment 4: SEQ ID NO. 4: ELNMDNILVEIK, respectively;
peptide fragment 12: SEQ ID NO. 12: ISGAGTGFGSR, respectively;
peptide fragment 25: SEQ ID No. 25: GEPGPPGEAGAAGPAGNPGADGQPGAK, respectively;
(2) peptide fragments SEQ ID No.31, 34, 36 are mule-hide-source and horse-hide-source shared polypeptides:
peptide fragment 31: SEQ ID NO. 31: GEPGGAGPVGPPGER, respectively;
peptide fragment 34: SEQ ID No. 34: GLLESEDGKLPRR, respectively;
peptide fragment 36: SEQ ID NO. 36: GLLESEDGK, respectively;
the detection is to detect whether the donkey-hide gelatin product sample contains mule skin-derived components, if the mule skin-derived components appear in the sample:
1) when mass spectrum detection spectrograms of the polypeptides shown in SEQ ID NO.31, 34 and 36 are carried out; and is
2) When mass spectrum detection spectrograms of the polypeptides shown in SEQ ID NO.4, 12 and 25 are carried out; and is
3) Detecting the absence of the polypeptide specific to the equine cortex component alone;
the existence of mule skin-derived components in the sample can be judged.
2. A detection method for detecting whether mule skin-derived components are contained in donkey-hide gelatin and products containing the donkey-hide gelatin comprises the following steps:
step (1), performing mass spectrum pretreatment on a sample to be detected to obtain a polypeptide filtrate to be detected:
step (2), detecting the polypeptide filtrate obtained in the step (1) by mass spectrometry, and detecting whether a sample to be detected contains a mass spectrum peak of the peptide fragment of claim 1;
as in the sample described:
1) when mass spectrum detection spectrograms of the polypeptides shown in SEQ ID NO.31, 34 and 36 are carried out; and is
2) When mass spectrum detection spectrograms of the polypeptides shown in SEQ ID NO.4, 12 and 25 are carried out; and is
3) Detecting the absence of the polypeptide specific to the equine cortex component alone;
judging the existence of mule skin-derived components in the sample;
the step (1) of performing mass spectrum pretreatment on a sample to be detected to obtain a polypeptide filtrate to be detected comprises the following steps:
1) homogenizing a sample to be tested into powder, weighing 0.1-0.5 g of donkey-hide gelatin sample or 1.0-2.0g of donkey-hide gelatin-containing product, and adding 1% NH4HCO3Carrying out ultrasonic treatment on the solution for 10-30min to completely dissolve the sample, and fixing the volume to 50 mL;
2) filtering the solution through a 0.22 mu m filter membrane to obtain filtrate;
3) adding 10-20 muL of Trypsin enzyme solution of 1 mug/muL into 100 muL-200 muL of the filtrate, performing enzymolysis at 37 ℃ for 16-18 hours, and waiting for on-machine detection;
step (2) the method of examining the polypeptide filtrate obtained in step (1) by mass spectrometry is the following method A or method B:
the method A comprises the following steps: detecting whether the polypeptide filtrate contains a mass spectrum peak of the peptide fragment of claim 1 by using AB SCIEX tripleTOF 5605600 mass spectrum,
the computer-operating parameters are as follows:
mobile phase A: 0.05-0.2% formic acid-acetonitrile solution, mobile phase B: 0.05 to 0.2% formic acid-water,
flow rate: 0.1 to 0.5mL/min,
TOF scan range: 100-3000Da of the total weight of the material,
positive ion reaction mode, GS 1: 35, GS 2: 45, Curtain Gas: 35, ISVF: 5500, TEM: 500, DP: 100, CE: 10;
the method B comprises the following steps: detecting whether the polypeptide filtrate contains a mass spectrum peak of the peptide fragment of claim 1 by AB SCIEX triple quadrupole mass spectrometry,
the computer-operating parameters are as follows:
mobile phase A: 0.05-0.2% formic acid-acetonitrile, mobile phase B: 0.05 to 0.2% formic acid-water,
flow rate: 0.1 to 0.5mL/min,
electrospray ion source, positive ion reaction mode, detection mode: MRM, spray voltage: 5500V, ion transfer tube temperature: 475 ℃; sheath gas pressure: 40; auxiliary gas pressure: 6.
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