CN106589063B - Donkey-derived characteristic peptides and application process thereof in qualitative detection of donkey skin and donkey-hide gelatin - Google Patents
Donkey-derived characteristic peptides and application process thereof in qualitative detection of donkey skin and donkey-hide gelatin Download PDFInfo
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Abstract
The invention discloses a group of donkey-derived characteristic peptides and an application process thereof in qualitative detection of donkey skin and donkey-hide gelatin, the donkey-derived characteristic peptides comprise five donkey-derived characteristic peptides, and the application process of the group of donkey-derived characteristic peptides in the aspect of donkey skin identification comprises the following steps: the method comprises the following steps: removing hair, subcutaneous fat and impurities from the skin sample; step two: carrying out enzymolysis by adopting trypsin: step three: carrying out liquid chromatography-mass spectrometry detection; step four: and carrying out multi-reaction monitoring by adopting an electrospray positive ion mode. An application process of a group of donkey-derived characteristic peptides in the aspect of donkey-hide gelatin identification comprises the following steps: the method comprises the following steps: carrying out enzymolysis by adopting trypsin; step two: carrying out liquid chromatography-mass spectrometry detection; step three: and carrying out multi-reaction monitoring by adopting an electrospray positive ion mode. The method has the beneficial effects that a group of donkey-derived characteristic peptides are obtained by comparing the results of enzymolysis of different species of collagen, and the characteristic polypeptide sequence of donkey is applied to qualitative detection of donkey skin and donkey hide gelatin.
Description
Technical Field
The invention relates to application of biological materials, in particular to a group of donkey-derived characteristic peptides and an application process in qualitative detection of donkey skin and donkey-hide gelatin thereof.
Background
The donkey-hide gelatin is prepared by decocting donkey hide, has multiple functions of nourishing yin, enriching blood and the like, is popular with consumers, and the annual output value of a series of products such as donkey-hide gelatin and related medicaments, foods, health-care foods and the like of the donkey-hide gelatin exceeds 300 hundred million at present. Due to the large demand of donkey-hide gelatin, the supply of donkey hide is small. Some illegal enterprises do not use donkey skin under the temptation of huge benefits, and use low-price horse skin, cow skin and pig skin as raw materials to produce donkey-hide gelatin, so that the fake gelatin does not contain donkey skin source components, and therefore, the method for detecting donkey source components in the donkey-hide gelatin with strong specificity and high sensitivity is very important to find.
Disclosure of Invention
The invention aims to solve the problems and designs a group of donkey-derived characteristic peptides and an application process thereof in qualitative detection of donkey skin and donkey-hide gelatin.
The technical scheme of the invention for realizing the aim is that a group of donkey-derived characteristic peptides and an application process thereof in qualitative detection of donkey skin and donkey-hide gelatin, and a group of donkey-derived characteristic peptides comprise five donkey-derived characteristic peptides which are respectively donkey-derived characteristic peptide 1 GPTGEPGKPGDK; donkey-derived characteristic peptide 2 GATGPAGVR; donkey-derived characteristic peptide 3 GEAGPQGAR; donkey-derived characteristic peptide 4 GEIGNPGR; ass derived signature peptide 5GEIGNPGR (P hydroxylated).
The donkey-derived characteristic peptide 1 is specific to horses, cattle, sheep and pigs by donkey, the donkey-derived characteristic peptide 2 is specific to horses and pigs by donkey, the donkey-derived characteristic peptide 3 and the donkey-derived characteristic peptide 4 are specific to cattle, sheep and pigs by donkey, and the donkey-derived characteristic peptide 5 is specific to cattle and sheep by donkey.
An application process of a group of donkey-derived characteristic peptides in donkey skin identification comprises the following steps:
the method comprises the following steps: removing hair, subcutaneous fat and impurities from the skin sample;
step two: carrying out enzymolysis by adopting trypsin: weighing a skin sample, degreasing the skin sample by using acetone, adding ultrapure water to dissolve the skin, after freeze drying, weighing 1.5mg of the skin sample, adding deionized water to prepare a protein sample with a certain concentration, adding a modified buffer solution, performing ultrafiltration substitution to obtain an ammonium bicarbonate buffer solution, adding trypsin, performing warm bath enzymolysis at 37 ℃, centrifuging by using an ultrafiltration tube, and collecting filtrate;
step three: carrying out LC-MS detection: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler; taking 0.1% formic acid water solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution: 0 → 5min, mobile phase A95%; 5 → 8min, 95% → 92% of mobile phase A; 8 → 10min, 92% → 50% of mobile phase A; 10 → 12min, mobile phase A50%; 12 → 12.1min, mobile phase A50% → 95%; 12.1 → 15min, 95% of mobile phase A and 0.3ml/min of flow rate;
step four: the method adopts an electrospray positive ion mode to carry out multi-reaction monitoring, donkey skin can be distinguished from donkey skin, horse skin, cow skin, sheep skin and pig skin according to detection results, and if five characteristic peptides are detected completely, the skin sample is donkey skin: selecting m/z380.7 → 374.82,493.56 for donkey-derived characteristic peptide 1; the donkey-derived characteristic peptide 2 is selected from m/z393.24 → 384.42,499.3; the donkey-derived characteristic peptide 3 is selected from m/z421.71 → 585.17,656.26; the donkey-derived characteristic peptide 4 is selected from m/z400.26 → 500.2,613.27; the donkey-derived characteristic peptide 5 is selected from m/z408.26 → 345.18,516.22; MRM scans are performed as ion pairs.
In step two, the denaturation buffer is 0.5M Tris-HCl, 2.75mM EDTA, 0.5M guanidine hydrochloride, pH 8.0.
In the fourth step, the electrospray positive ion mode uses triple quadrupole mass spectrometry as a detector, and the electrospray ion source is ESI +.
An application process of a group of donkey-derived characteristic peptides in the aspect of donkey-hide gelatin identification comprises the following steps:
the method comprises the following steps: carrying out enzymolysis by adopting trypsin: weighing 0.2g of glue sample, dissolving in 100ml of 1% ammonium bicarbonate buffer solution, adding trypsin, and performing enzymolysis at 37 ℃; centrifuging by an ultrafiltration tube, and collecting filtrate;
step two: carrying out LC-MS detection: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler; taking 0.1% formic acid water solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution: 0 → 5min, mobile phase A95%; 5 → 8min, 95% → 92% of mobile phase A; 8 → 10min, 92% → 50% of mobile phase A; 10 → 12min, mobile phase A50%; 12 → 12.1min, mobile phase A50% → 95%; 12.1 → 15min, 95% of mobile phase A and 0.3ml/min of flow rate;
step three: carrying out multi-reaction monitoring in an electrospray positive ion mode, and distinguishing donkey-hide gelatin from donkey-hide gelatin, horse hide gelatin, cow hide gelatin, sheep hide gelatin and pig hide gelatin according to a detection result: if all five characteristic peptides are detected, the sample is donkey-hide gelatin: selecting m/z380.7 → 374.82,493.56 for donkey-derived characteristic peptide 1; the donkey-derived characteristic peptide 2 is selected from m/z393.24 → 384.42,499.3; the donkey-derived characteristic peptide 3 is selected from m/z421.71 → 585.17,656.26; the donkey-derived characteristic peptide 4 is selected from m/z400.26 → 500.2,613.27; the donkey-derived characteristic peptide 5 is selected from m/z408.26 → 345.18,516.22; performing MRM scanning as ion pairs;
in the third step, the electrospray positive ion mode takes triple quadrupole mass spectrometry as a detector, and the electrospray ion source is ESI +.
The application process of the qualitative detection of the donkey-derived characteristic peptides and the donkey skin and the donkey-hide gelatin thereof manufactured by the technical scheme of the invention simulates the result of trypsin enzymolysis of collagen of different species through the Peptidemass function provided by Uniprot, obtains the characteristic polypeptide sequence of the donkey relative to other species by comparing the collagen sequence of the donkey with the sequence of other species collagen, and applies the characteristic polypeptide sequence to the qualitative detection of the donkey skin and the donkey-hide gelatin.
Drawings
FIG. 1 is a schematic structural diagram of the application process of a group of donkey-derived characteristic peptides in donkey skin identification according to the invention;
FIG. 2 is a schematic structural diagram of the application process of a group of donkey-derived characteristic peptides in donkey-hide gelatin identification according to the present invention;
FIG. 3 is a liquid phase mass spectrometric detection of donkey-derived signature peptide 1 of the present invention;
FIG. 4 is a liquid phase mass spectrometric detection of donkey-derived signature peptide 2 of the present invention;
FIG. 5 is a liquid phase mass spectrometric detection of donkey-derived signature peptide 3 according to the present invention;
FIG. 6 is a liquid phase mass spectrometric detection of donkey-derived signature peptide 4 of the present invention;
FIG. 7 is a liquid phase mass spectrometric detection of donkey-derived signature peptide 5 of the present invention;
Detailed Description
The present invention is described in detail with reference to the accompanying drawings, as shown in fig. 1-7, a group of donkey-derived characteristic peptides and their application process in qualitative detection of donkey skin and donkey-hide gelatin, a group of donkey-derived characteristic peptides including five donkey-derived characteristic peptides, wherein the five donkey-derived characteristic peptides are respectively donkey-derived characteristic peptide 1 GPTGEPGKPGDK; donkey-derived characteristic peptide 2 GATGPAGVR; donkey-derived characteristic peptide 3 GEAGPQGAR; donkey-derived characteristic peptide 4 GEIGNPGR; donkey-derived characteristic peptide 5GEIGNPGR (P-hydroxylation); the donkey-derived characteristic peptide 1 is specific to the donkey relative to the horse, the cow, the sheep and the pig, the donkey-derived characteristic peptide 2 is specific to the donkey relative to the horse and the pig, the donkey-derived characteristic peptide 3 and the donkey-derived characteristic peptide 4 are specific to the donkey relative to the cow, the sheep and the pig, and the donkey-derived characteristic peptide 5 is specific to the donkey relative to the cow and the sheep; an application process of a group of donkey-derived characteristic peptides in donkey skin identification comprises the following steps: the method comprises the following steps: removing hair, subcutaneous fat and impurities from the skin sample; step two: carrying out enzymolysis by adopting trypsin: weighing a skin sample, degreasing the skin sample by using acetone, adding ultrapure water to dissolve the skin, after freeze drying, weighing 1.5mg of the skin sample, adding deionized water to prepare a protein sample with a certain concentration, adding a modified buffer solution, performing ultrafiltration substitution to obtain an ammonium bicarbonate buffer solution, adding trypsin, performing warm bath enzymolysis at 37 ℃, centrifuging by using an ultrafiltration tube, and collecting filtrate; step three: carrying out LC-MS detection: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler; taking 0.1% formic acid water solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution: 0 → 5min, mobile phase A95%; 5 → 8min, 95% → 92% of mobile phase A; 8 → 10min, 92% → 50% of mobile phase A; 10 → 12min, mobile phase A50%; 12 → 12.1min, mobile phase A50% → 95%; 12.1 → 15min, 95% of mobile phase A and 0.3ml/min of flow rate; step four: the method adopts an electrospray positive ion mode to carry out multi-reaction monitoring, donkey skin can be distinguished from donkey skin, horse skin, cow skin, sheep skin and pig skin according to detection results, and if five characteristic peptides are detected completely, the skin sample is donkey skin: selecting m/z380.7 → 374.82,493.56 for donkey-derived characteristic peptide 1; the donkey-derived characteristic peptide 2 is selected from m/z393.24 → 384.42,499.3; the donkey-derived characteristic peptide 3 is selected from m/z421.71 → 585.17,656.26; the donkey-derived characteristic peptide 4 is selected from m/z400.26 → 500.2,613.27; the donkey-derived characteristic peptide 5 is selected from m/z408.26 → 345.18,516.22; performing MRM scanning as ion pairs; in the second step, the denaturation buffer is 0.5M Tris-HCl, 2.75mM EDTA, 0.5M guanidine hydrochloride, pH8.0 buffer; in the fourth step, the electrospray positive ion mode takes triple quadrupole mass spectrometry as a detector, and the electrospray ion source is ESI +; an application process of a group of donkey-derived characteristic peptides in the aspect of donkey-hide gelatin identification comprises the following steps: the method comprises the following steps: carrying out enzymolysis by adopting trypsin: weighing 0.2g of glue sample, dissolving in 100ml of 1% ammonium bicarbonate buffer solution, adding trypsin, and performing enzymolysis at 37 ℃; centrifuging by an ultrafiltration tube, and collecting filtrate; step two: carrying out LC-MS detection: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler; taking 0.1% formic acid water solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution: 0 → 5min, mobile phase A95%; 5 → 8min, 95% → 92% of mobile phase A; 8 → 10min, 92% → 50% of mobile phase A; 10 → 12min, mobile phase A50%; 12 → 12.1min, mobile phase A50% → 95%; 12.1 → 15min, 95% of mobile phase A and 0.3ml/min of flow rate; step three: carrying out multi-reaction monitoring in an electrospray positive ion mode, and distinguishing donkey-hide gelatin from donkey-hide gelatin, horse hide gelatin, cow hide gelatin, sheep hide gelatin and pig hide gelatin according to a detection result: if all five characteristic peptides are detected, the sample is donkey-hide gelatin: selecting m/z380.7 → 374.82,493.56 for donkey-derived characteristic peptide 1; the donkey-derived characteristic peptide 2 is selected from m/z393.24 → 384.42,499.3; the donkey-derived characteristic peptide 3 is selected from m/z421.71 → 585.17,656.26; the donkey-derived characteristic peptide 4 is selected from m/z400.26 → 500.2,613.27; the donkey-derived characteristic peptide 5 is selected from m/z408.26 → 345.18,516.22; performing MRM scanning as ion pairs; in the third step, the electrospray positive ion mode takes triple quadrupole mass spectrometry as a detector, and the electrospray ion source is ESI +.
The embodiment is characterized in that a group of donkey-derived characteristic peptides comprises five donkey-derived characteristic peptides, wherein the five donkey-derived characteristic peptides are donkey-derived characteristic peptides 1GPTGEPGKPGDK respectively; donkey-derived characteristic peptide 2 GATGPAGVR; donkey-derived characteristic peptide 3 GEAGPQGAR; donkey-derived characteristic peptide 4 GEIGNPGR; donkey-derived characteristic peptide 5GEIGNPGR (P hydroxylation), and an application process of a group of donkey-derived characteristic peptides in donkey skin identification, comprises the following steps: the method comprises the following steps: removing hair, subcutaneous fat and impurities from the skin sample; step two: carrying out enzymolysis by adopting trypsin; step three: carrying out liquid chromatography-mass spectrometry detection; step four: carrying out multi-reaction monitoring by adopting an electrospray positive ion mode, distinguishing donkey skins from donkey skins, horse skins, cowhides, sheep skins and pig skins according to detection results, wherein if all five characteristic peptides are detected, the skin sample is donkey skins, and the application process of a group of donkey-derived characteristic peptides in the aspect of donkey-hide identification comprises the following steps: the method comprises the following steps: carrying out enzymolysis by adopting trypsin; step two: carrying out liquid chromatography-mass spectrometry detection; step three: carrying out multi-reaction monitoring in an electrospray positive ion mode, and distinguishing donkey-hide gelatin from donkey-hide gelatin, horse hide gelatin, cow hide gelatin, sheep hide gelatin and pig hide gelatin according to a detection result: if all five characteristic peptides are detected, the sample is donkey-hide gelatin, the result of trypsin enzymolysis of collagen of different species is simulated through the Peptidomass function provided by Unit, the characteristic polypeptide sequence of donkey relative to other species is obtained by comparing the collagen sequence of donkey with the sequence of other species collagen, and the characteristic polypeptide sequence is applied to qualitative detection of donkey skin and donkey-hide gelatin.
In this embodiment, ammonium bicarbonate, trypsin, and acetonitrile were used in the assay from Sigma and formic acid from Merck. All reagents were chromatographically pure. The application process of a group of donkey-derived characteristic peptides in the aspect of donkey skin identification comprises the following steps: the method comprises the following steps: removing hair, subcutaneous fat and impurities from the skin sample; step two: carrying out enzymolysis by adopting trypsin: weighing a skin sample, degreasing by using acetone, adding ultrapure water to dissolve the skin, after freeze drying, weighing 1.5mg, adding deionized water to prepare a protein sample with a certain concentration, adding a modified buffer solution, performing ultrafiltration replacement to obtain a ammonium bicarbonate buffer solution, adding trypsin, performing warm bath enzymolysis at 37 ℃, centrifuging by using an ultrafiltration tube, and collecting filtrate; step three: carrying out LC-MS detection: the detection conditions of HPLC-TQ-SMS/MS are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler; taking 0.1% formic acid water solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution: 0 → 5min, mobile phase A95%; 5 → 8min, 95% → 92% of mobile phase A; 8 → 10min, 92% → 50% of mobile phase A; 10 → 12min, mobile phase A50%; 12 → 12.1min, mobile phase A50% → 95%; 12.1 → 15min, 95% of mobile phase A and 0.3ml/min of flow rate; step four: the method adopts an electrospray positive ion mode to carry out multi-reaction monitoring, donkey skin can be distinguished from donkey skin, horse skin, cow skin, sheep skin and pig skin according to detection results, and if five characteristic peptides are detected completely, the skin sample is donkey skin: selecting m/z380.7 → 374.82,493.56 for donkey-derived characteristic peptide 1; the donkey-derived characteristic peptide 2 is selected from m/z393.24 → 384.42,499.3; the donkey-derived characteristic peptide 3 is selected from m/z421.71 → 585.17,656.26; the donkey-derived characteristic peptide 4 is selected from m/z400.26 → 500.2,613.27; the donkey-derived characteristic peptide 5 is selected from m/z408.26 → 345.18,516.22; MRM scans are performed as ion pairs.
In this embodiment, a ThermoScientific Fusion-Orbitrap high resolution mass spectrometer is used, and the ion source is a Nanospray Flex source. Collagen is the main protein in the dermis of animals, so the invention selects proper polypeptide in the collagen as the characteristic polypeptide of donkey skin collagen. Simulating by utilizing a PeptideMess function provided by Uniport to obtain a result of trypsin enzymolysis of collagen of donkey skin, horse skin, cow skin, sheep skin and pig skin; the sequences of collagen of donkey hide, horse hide, cow hide, sheep hide and pig hide were aligned by using molecular biology software MEGA 5.0. In order to verify the characteristic peptide of the donkey skin, horse skin, cow skin, sheep skin and pig skin are selected, and according to the sample pretreatment method and the EASY-nLC1000-Orbitrap Fusion LC-MS, the detection result shows that the molecular weight and the secondary mass spectrum of the characteristic peptide are consistent with the theoretical value.
In this embodiment, the application process of a group of donkey-derived characteristic peptides in the aspect of donkey-hide gelatin identification includes the following steps: the method comprises the following steps: carrying out enzymolysis by adopting trypsin: weighing 0.2g of glue sample, dissolving in 100ml of 1% ammonium bicarbonate buffer solution, adding trypsin, and performing enzymolysis at 37 ℃; centrifuging by an ultrafiltration tube, and collecting filtrate; step two: carrying out LC-MS detection: conditions for HPLC-TQ-SMS/MS were as follows: the adopted chromatographic column takes octadecylsilane chemically bonded silica as a filler (the inner diameter of the chromatographic column is 2.1 mm); taking 0.1% formic acid water solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution: 0 → 5min, mobile phase A95%; 5 → 8min, 95% → 92% of mobile phase A; 8 → 10min, 92% → 50% of mobile phase A; 10 → 12min, mobile phase A50%; 12 → 12.1min, mobile phase A50% → 95%; 12.1 → 15min, 95% of mobile phase A and 0.3ml/min of flow rate; step three: the multi-reaction monitoring is carried out in an electrospray positive ion mode, the electrospray positive ion mode takes triple quadrupole mass spectrum as a detector, an electrospray ion source is ESI +, and donkey-hide gelatin can be distinguished from donkey-hide gelatin, horse hide gelatin, cow hide gelatin, sheep hide gelatin and pig hide gelatin according to a detection result: if all five characteristic peptides are detected, the sample is donkey-hide gelatin: the donkey-derived characteristic peptide 1 was selected to be m/z380.7 → 374.82,493.56, as shown in FIG. 3, A: extracting an ion flow; b: primary mass spectrum; c: a secondary mass spectrum; (ii) a The donkey-derived signature peptide 2 was selected m/z393.24 → 384.42,499.3 as shown in fig. 4, a: extracting an ion flow; b: primary mass spectrum; c: a secondary mass spectrum; the donkey-derived signature peptide 3 was selected m/z421.71 → 585.17,656.26, as shown in fig. 5, a: extracting an ion flow; b: primary mass spectrum; c: a secondary mass spectrum; the donkey-derived signature peptide 4 was selected m/z400.26 → 500.2,613.27 as shown in fig. 6, a: extracting an ion flow; b: primary mass spectrum; c: a secondary mass spectrum; the donkey-derived signature peptide 5 was selected m/z408.26 → 345.18,516.22, as shown in fig. 7, a: extracting an ion flow; b: primary mass spectrum; c: a secondary mass spectrum; MRM scans are performed as ion pairs.
In this embodiment, example 1, identification of a set of donkey-derived signature peptides:
collagen is the main protein in the dermis of animals, so the invention selects proper polypeptide in the collagen as the characteristic polypeptide of donkey skin collagen. Simulating by utilizing a PeptideMess function provided by Uniport to obtain a result of trypsin enzymolysis of collagen of donkey skin, horse skin, cow skin, sheep skin and pig skin; and (3) comparing the sequences of collagen of donkey skin, horse skin, cow skin, sheep skin and pig skin by using molecular biology software MEGA5.0 to obtain a theoretical characteristic peptide sequence. In order to verify the donkey hide characteristic peptide, donkey hide, horse hide, cow hide, sheep hide, pig hide were selected according to the sample pretreatment method described above and the EASY-nLC1000-Orbitrap Fusion LC MS/MS analysis method. The detection results are shown in FIGS. 3-7, and the molecular weight and the secondary mass spectrum of the characteristic peptide are consistent with theoretical values.
Wherein, the detection conditions of the EASY-nLC1000-Orbitrap Fusion LC-MS method are as follows: first, separation was performed using an EASY-nLC1000 nanoliter liquid phase system. Wherein a 0.1% aqueous formic acid solution with mobile phase A of 2% acetonitrile and a 0.1% aqueous formic acid solution with mobile phase B of 98% acetonitrile were used as detection reagents. The device can be set by a worker, the elution gradient is set to be 0 → 15min in the first stage, the flow rate is 300 nL.min < -1 >, the mobile phase A is 100%, 15 → 70min in the second stage, the flow rate is 300 nL.min < -1 >, the mobile phase A is 93% → 78%, the third stage is 70 → 90min, the flow rate is 300 nL.min < -1 >, the mobile phase A is 78% → 65%, the fourth stage is 90 → 95min, the flow rate is 300 nL.min < -1 >, the mobile phase A is 65% → 10%, the fifth stage is 95 → 105min, the flow rate is 450 nL.min < -1 >, the mobile phase A is 10%, and the sample injection amount is 1 μ l. Secondly, 0.2mm multiplied by 3.5cm (5 mu m particle size) Repsil-Pur C18-AQ Trap column is adopted for desalting and enriching work, wherein the adopted instrument is a Repsil-Pur C18-AQ nanoliter analytical column separation device which is manufactured by a laboratory and has the particle size of 75 mu m multiplied by 25cm (3 mu m). Thirdly, a Fusion-Orbitrap high-resolution mass spectrometer of Thermo Scientific is adopted for detection, wherein the ion source is a Nanospray Flex nano source. The analysis is carried out in a positive ion mode, the spraying voltage is 2.1kV, the temperature of an ion transmission capillary is 275 ℃, and the S-Lens transmission efficiency is set to be 60 percent. The primary mass spectrum uses Orbitrap as the mass analyzer with a resolution of 60,000(m/z 400) and an acquisition range of 350-1,650 Th. The secondary mass spectrum adopts an ion trap as a mass analyzer, adopts a Rapid Scan mode for scanning, utilizes a Top20 data dependence mode for parent ion selection, adopts a CID mode for fragmentation, and sets the fragmentation energy NCE to be 35%, thereby completing the detection work.
In this embodiment, in example 2, the MRM detection conditions of the donkey-derived characteristic peptide are as follows:
the characteristic peptides are optimized by a triple quadrupole liquid chromatography-mass spectrometry method, and the optimized MRM parameters are shown in Table 1:
TABLE 1 MRM detection conditions for donkey skin characteristic peptide
| Composition (I) | Parent ion (m/z) | Ionic acid (m/z) | Taper hole voltage (v) | Collision energy (ev) |
| Ass-derived characteristic peptide 1 | 380.7 | 374.82 | 20 | 10 |
| Ass-derived characteristic peptide 1 | 380.7 | 493.56 | 20 | 10 |
| Ass-derived characteristic peptide 2 | 393.24 | 384.42 | 15 | 10 |
| Ass-derived characteristic peptide 2 | 393.24 | 499.3 | 15 | 16 |
| Ass-derived |
421.71 | 585.17 | 18 | 16 |
| Ass-derived |
421.71 | 656.26 | 18 | 16 |
| Ass-derived characteristic peptide 4 | 400.26 | 500.2 | 16 | 15 |
| Ass-derived characteristic peptide 4 | 400.26 | 613.27 | 16 | 15 |
| Ass-derived |
408.26 | 345.18 | 18 | 15 |
| Ass-derived |
408.26 | 516.22 | 18 | 15 |
Wherein, the triple quadrupole liquid chromatography-mass spectrometry is the same as the HPLC-TQ-SMS/MS liquid chromatography-mass spectrometry detection method in the claims.
The technical solutions described above only represent the preferred technical solutions of the present invention, and some possible modifications to some parts of the technical solutions by those skilled in the art all represent the principles of the present invention, and fall within the protection scope of the present invention.
Claims (6)
1. The donkey-derived characteristic peptides are characterized by comprising five donkey-derived characteristic peptides, wherein the five donkey-derived characteristic peptides are donkey-derived characteristic peptides 1GPTGEPGKPGDK respectively; donkey-derived characteristic peptide 2 GATGPAGVR; donkey-derived characteristic peptide 3 GEAGPQGAR; donkey-derived characteristic peptide 4 GEIGNPGR; donkey-derived characteristic peptide 5GEIGNPGR, P hydroxylation in the donkey-derived characteristic peptide 5, donkey-derived characteristic peptide 1 is specific to horses, cattle, sheep and pigs, donkey-derived characteristic peptide 2 is specific to horses and pigs, donkey-derived characteristic peptide 3 and donkey-derived characteristic 4 are specific to cattle, sheep and pigs, and donkey-derived characteristic peptide 5 is specific to cattle and sheep.
2. Use of a panel of donkey-derived signature peptides according to claim 1 in donkey skin identification, characterized in that it comprises the following steps:
the method comprises the following steps: removing hair, subcutaneous fat and impurities from the skin sample;
step two: carrying out enzymolysis by adopting trypsin: weighing a skin sample, degreasing the skin sample by using acetone, adding ultrapure water to dissolve the skin, after freeze drying, weighing 1.5mg of the skin sample, adding deionized water to prepare a protein sample with a certain concentration, adding a modified buffer solution, performing ultrafiltration substitution to obtain an ammonium bicarbonate buffer solution, adding trypsin, performing warm bath enzymolysis at 37 ℃, centrifuging by using an ultrafiltration tube, and collecting filtrate;
step three: carrying out LC-MS detection: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler; taking 0.1% formic acid water solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution: 0 → 5min, mobile phase A95%; 5 → 8min, 95% → 92% of mobile phase A; 8 → 10min, 92% → 50% of mobile phase A; 10 → 12min, mobile phase A50%; 12 → 12.1min, mobile phase A50% → 95%; 12.1 → 15min, 95% of mobile phase A and 0.3ml/min of flow rate;
step four: the method adopts an electrospray positive ion mode to carry out multi-reaction monitoring, donkey skin can be distinguished from donkey skin, horse skin, cow skin, sheep skin and pig skin according to detection results, and if five characteristic peptides are detected completely, the skin sample is donkey skin: selecting m/z380.7 → 374.82,493.56 for donkey-derived characteristic peptide 1; the donkey-derived characteristic peptide 2 is selected from m/z393.24 → 384.42,499.3; the donkey-derived characteristic peptide 3 is selected from m/z421.71 → 585.17,656.26; the donkey-derived characteristic peptide 4 is selected from m/z400.26 → 500.2,613.27; the donkey-derived characteristic peptide 5 is selected from m/z408.26 → 345.18,516.22; MRM scans are performed as ion pairs.
3. The use of a set of donkey-derived signature peptides according to claim 2 in donkey skin identification, wherein in step two, the denaturation buffer is 0.5M Tris-HCl, 2.75mM EDTA, 0.5M guanidine hydrochloride, pH 8.0.
4. The use of a set of donkey-derived signature peptides according to claim 2 in donkey skin identification, characterized in that in step four, the electrospray positive ion mode uses triple quadrupole mass spectrometry as a detector, and the electrospray ion source is ESI +.
5. The use of the donkey-derived signature peptide of claim 1 in donkey-hide gelatin identification, comprising the following steps:
the method comprises the following steps: carrying out enzymolysis by adopting trypsin: weighing 0.2g of glue sample, dissolving in 100ml of 1% ammonium bicarbonate buffer solution, adding trypsin, and performing enzymolysis at 37 ℃; centrifuging by an ultrafiltration tube, and collecting filtrate;
step two: carrying out LC-MS detection: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler; taking 0.1% formic acid water solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution: 0 → 5min, mobile phase A95%; 5 → 8min, 95% → 92% of mobile phase A; 8 → 10min, 92% → 50% of mobile phase A; 10 → 12min, mobile phase A50%; 12 → 12.1min, mobile phase A50% → 95%; 12.1 → 15min, 95% of mobile phase A and 0.3ml/min of flow rate;
step three: carrying out multi-reaction monitoring in an electrospray positive ion mode, and distinguishing donkey-hide gelatin from donkey-hide gelatin, horse hide gelatin, cow hide gelatin, sheep hide gelatin and pig hide gelatin according to a detection result: if all five characteristic peptides are detected, the sample is donkey-hide gelatin: selecting m/z380.7 → 374.82,493.56 for donkey-derived characteristic peptide 1; the donkey-derived characteristic peptide 2 is selected from m/z393.24 → 384.42,499.3; the donkey-derived characteristic peptide 3 is selected from m/z421.71 → 585.17,656.26; the donkey-derived characteristic peptide 4 is selected from m/z400.26 → 500.2,613.27; the donkey-derived characteristic peptide 5 is selected from m/z408.26 → 345.18,516.22; MRM scans are performed as ion pairs.
6. The use of a set of donkey-derived signature peptides according to claim 5 in the identification of donkey-hide gelatin, wherein in step three, the electrospray positive ion mode uses triple quadrupole mass spectrometry as a detector, and the electrospray ion source is ESI +.
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| CN110470566A (en) * | 2019-09-02 | 2019-11-19 | 山东省食品药品检验研究院 | A kind of colla corii asini cake class product determination of fat method |
| CN110824083B (en) * | 2019-11-06 | 2022-04-12 | 东阿阿胶股份有限公司 | Application of donkey-bone glue characteristic polypeptide in detection of donkey-bone glue components in animal skin glue and products thereof |
| CN111855863A (en) * | 2020-08-14 | 2020-10-30 | 江西师范大学 | Method for identifying gelatin source in donkey-hide gelatin product |
| CN112763644B (en) * | 2020-12-17 | 2024-02-06 | 中国检验检疫科学研究院 | Characteristic peptide composition for detecting milk powder doped in donkey milk powder and detection method |
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