CN106589063A - Donkey-source characteristic peptide group and application process thereof in qualitative detection of donkey skin and donkey-hide gelatin - Google Patents
Donkey-source characteristic peptide group and application process thereof in qualitative detection of donkey skin and donkey-hide gelatin Download PDFInfo
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- CN106589063A CN106589063A CN201611148596.5A CN201611148596A CN106589063A CN 106589063 A CN106589063 A CN 106589063A CN 201611148596 A CN201611148596 A CN 201611148596A CN 106589063 A CN106589063 A CN 106589063A
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- C—CHEMISTRY; METALLURGY
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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Abstract
The invention discloses a donkey-source characteristic peptide group and an application process thereof in qualitative detection of donkey skin and donkey-hide gelatin. The donkey-source characteristic peptide group comprises five donkey-source characteristic peptides. The application process of the donkey-source characteristic peptide group in donkey skin identification includes the steps: first, removing hair, subcutaneous fat and impurities of a skin sample; second, performing enzymolysis by the aid of trypsase; third, performing detection for liquid chromatography-mass spectrometry; fourth, performing multi-reaction monitoring in an electrospray positive ion mode. The application process of the donkey-source characteristic peptide group in donkey-hide gelatin identification includes the steps: first, performing enzymolysis by the aid of the trypsase; second, performing detection for liquid chromatography-mass spectrometry; third, performing multi-reaction monitoring in the electrospray positive ion mode. The donkey-source characteristic peptide group has the advantages that enzymolysis results of collagen of different species are compared to obtain the donkey-source characteristic peptide group, and a donkey characteristic peptide sequence is applied to qualitative detection of the donkey skin and the donkey-hide gelatin.
Description
Technical field
The present invention relates to group donkey source property feature peptide of biomaterial application, particularly and its donkey skin and Colla Corii Asini glue qualitative detection
In application flow.
Background technology
Colla Corii Asini is boiled by donkey skin and is formed, multiple with nourishing YIN and supplementing blood etc., deep to be welcome by consumers in general, current Colla Corii Asini and
The series of products annual value of production such as the related medicine of Colla Corii Asini, food, health food already exceed 30,000,000,000.Due to the demand of Colla Corii Asini it is big, donkey
The supply of skin is few.Some illegal enterprises do not use donkey skin under the temptation of huge interests, and use low-cost Corium Equi, cattle
Skin, Corii Sus domestica do raw material production Colla Corii Asini, cause these pseudo-gums without donkey skin derived components, therefore find that specificity is strong, sensitivity is high
In detection Colla Corii Asini, the method tool of donkey derived component is of great significance.
The content of the invention
The invention aims to solve the above problems, one group of donkey source property feature peptide is devised and its in donkey skin and Colla Corii Asini
Application flow in glue qualitative detection.
Realize that above-mentioned purpose the technical scheme is that, one group of donkey source property feature peptide and its qualitative in donkey skin and Colla Corii Asini glue
Application flow in detection, one group of donkey source property feature peptide, including five donkey source property feature peptides, five donkey sources property feature peptide point
Wei not donkey source property feature peptide 1GPTGEPGKPGDK；Donkey source property feature peptide 2GATGPAGVR；Donkey source property feature peptide 3GEAGPQGAR；
Donkey source property feature peptide 4GEIGNPGR；Donkey source property feature peptide 5GEIGNPGR (P hydroxylatings).
It is peculiar relative to horse, cattle, sheep, pig institute that the donkey source property feature peptide 1 is donkey, donkey source property feature peptide 2 be donkey relative to
Horse and pig institute are peculiar, and donkey source property feature peptide 3 and donkey source property 4 are that donkey is peculiar relative to cattle, sheep, pig institute, and donkey source property feature peptide 5 is donkey
It is peculiar relative to cattle, sheep institute.
One group of donkey source property feature peptide in terms of donkey skin discriminating in application flow, comprise the steps：
Step one：By skin sample unhairing, subcutaneous fat and impurity；
Step 2：Using trypsin digestion：Skin class sample is weighed, ungrease treatment is carried out using acetone, add ultra-pure water
Skin is dissolved, 1.5mg is weighed after lyophilization and is added deionized water to be made into certain density protein sample, add degeneration buffering
Liquid, carries out ultrafiltration and is replaced into ammonium hydrogencarbonate buffer, adds 37 DEG C of temperature bath enzymolysis of trypsin, super filter tube centrifugal filtration to collect filter
Liquid；
Step 3：Carry out LC-MS detection：The chromatographic column for adopting is with octadecylsilane chemically bonded silica as filler；With
0.1% aqueous formic acid is mobile phase A, with acetonitrile as Mobile phase B, gradient elution：0 → 5min, mobile phase A are 95%；5→
8min, mobile phase A are 95% → 92%；8 → 10min, mobile phase A are 92% → 50%；10 → 12min, mobile phase A is
50%；12 → 12.1min, mobile phase A are 50% → 95%；12.1 → 15min, mobile phase A are 95%, and flow velocity is 0.3ml/
min；
Step 4：Multiple-reaction monitoring is carried out using electron spray positive ion mode, can be from donkey skin, horse according to testing result
Donkey skin is distinguished in skin, Corii Bovis seu Bubali, Corii Caprae seu Oviss and Corii Sus domestica, if five feature peptides are all detected, the skin sample is donkey skin：Donkey source property feature
Peptide 1 selects m/z380.7 → 374.82,493.56；Donkey source property feature peptide 2 selects m/z393.24 → 384.42,499.3；Donkey source
Property feature peptide 3 select m/z421.71 → 585.17,656.26；Donkey source property feature peptide 4 selects m/z400.26 → 500.2,
613.27；Donkey source property feature peptide 5 selects m/z408.26 → 345.18,516.22；MRM scannings are carried out as ion pair.
In step 2, the denaturation buffer be 0.5MTris-HCl, 2.75mM ethylenediaminetetraacetic acid, 0.5M guanidine hydrochlorides,
The buffer of pH8.0.
In step 4, the electron spray positive ion mode using triple quadrupole bar mass spectrum as detector, described its electron spray
Ion source is ESI+.
One group of donkey source property feature peptide in terms of Colla Corii Asini discriminating in application flow, comprise the steps：
Step one：Using trypsin digestion：Weigh 0.2g glue class samples and be dissolved in 1% ammonium hydrogencarbonate buffer of 100ml,
Add 37 DEG C of temperature bath enzymolysis of trypsin；Filtrate is collected in super filter tube centrifugal filtration；
Step 2：Carry out LC-MS detection：The chromatographic column for adopting is with octadecylsilane chemically bonded silica as filler；With
0.1% aqueous formic acid is mobile phase A, with acetonitrile as Mobile phase B, gradient elution：0 → 5min, mobile phase A are 95%；5→
8min, mobile phase A are 95% → 92%；8 → 10min, mobile phase A are 92% → 50%；10 → 12min, mobile phase A is
50%；12 → 12.1min, mobile phase A are 50% → 95%；12.1 → 15min, mobile phase A are 95%, and flow velocity is 0.3ml/
min；
Step 3：Multiple-reaction monitoring is carried out using electron spray positive ion mode, can be from Colla Corii Asini, Corium Equi according to testing result
Colla Corii Asini is distinguished in glue, animal glue, Corii Caprae seu Oviss glue and pig skin gelatinum：If five feature peptide is all detected, the sample is Colla Corii Asini：Donkey source
Property feature peptide 1 select m/z380.7 → 374.82,493.56；Donkey source property feature peptide 2 selects m/z393.24 → 384.42,
499.3；Donkey source property feature peptide 3 selects m/z421.71 → 585.17,656.26；Donkey source property feature peptide 4 select m/z400.26 →
500.2,613.27；Donkey source property feature peptide 5 selects m/z408.26 → 345.18,516.22；MRM scannings are carried out as ion pair；
In step 3, the electron spray positive ion mode using triple quadrupole bar mass spectrum as detector, described its electron spray
Ion source is ESI+.
In the one group of donkey source property feature peptide made using technical scheme and its donkey skin and Colla Corii Asini glue qualitative detection
Application flow, the present invention by Uniprot offer Peptidemass functional simulation trypsin digestion different plant species collagens
The result of albumen, and donkey is obtained relative to which by the sequence alignment by the collagen sequences of donkey with other species collagen protein
The characteristic peptide sequence of his species, and apply it in the qualitative detection of donkey skin and Colla Corii Asini.
Description of the drawings
Fig. 1 be one group of donkey source of the present invention property feature peptide in terms of donkey skin discriminating in application flow structural representation
Figure；
Fig. 2 be one group of donkey source of the present invention property feature peptide in terms of Colla Corii Asini discriminating in application flow structural representation
Figure；
Fig. 3 is the liquid chromatography mass spectrometric detection figure of property feature peptide in donkey source of the present invention 1；
Fig. 4 is the liquid chromatography mass spectrometric detection figure of property feature peptide in donkey source of the present invention 2；
Fig. 5 is the liquid chromatography mass spectrometric detection figure of property feature peptide in donkey source of the present invention 3；
Fig. 6 is the liquid chromatography mass spectrometric detection figure of property feature peptide in donkey source of the present invention 4；
Fig. 7 is the liquid chromatography mass spectrometric detection figure of property feature peptide in donkey source of the present invention 5；
Specific embodiment
Below in conjunction with the accompanying drawings the present invention is specifically described, as shown in figs. 1-7, one group of donkey source property feature peptide and its in donkey
Application flow in skin and Colla Corii Asini glue qualitative detection, one group of donkey source property feature peptide, including five donkey source property feature peptides, described five
Donkey source property feature peptide is respectively donkey source property feature peptide 1GPTGEPGKPGDK；Donkey source property feature peptide 2GATGPAGVR；Donkey source property feature
Peptide 3GEAGPQGAR；Donkey source property feature peptide 4GEIGNPGR；Donkey source property feature peptide 5GEIGNPGR (P hydroxylatings)；The donkey source property
It is peculiar relative to horse, cattle, sheep, pig institute that feature peptide 1 is donkey, and donkey source property feature peptide 2 is donkey relative to horse and peculiar, the donkey source property of pig
It is peculiar relative to cattle, sheep, pig institute that feature peptide 3 and donkey source property 4 are donkeys, and donkey source property feature peptide 5 is that donkey is peculiar relative to cattle, sheep institute；
One group of donkey source property feature peptide in terms of donkey skin discriminating in application flow, comprise the steps：Step one：By skin sample unhairing, skin
Lower fat and impurity；Step 2：Using trypsin digestion：Skin class sample is weighed, ungrease treatment is carried out using acetone, add super
Skin is dissolved by pure water, is weighed 1.5mg and is added deionized water to be made into certain density protein sample, add degeneration to delay after lyophilization
Liquid is rushed, ultrafiltration is carried out and is replaced into ammonium hydrogencarbonate buffer, add 37 DEG C of temperature bath enzymolysis of trypsin, super filter tube centrifugal filtration to collect
Filtrate；Step 3：Carry out LC-MS detection：The chromatographic column for adopting is with octadecylsilane chemically bonded silica as filler；With
0.1% aqueous formic acid is mobile phase A, with acetonitrile as Mobile phase B, gradient elution：0 → 5min, mobile phase A are 95%；5→
8min, mobile phase A are 95% → 92%；8 → 10min, mobile phase A are 92% → 50%；10 → 12min, mobile phase A is
50%；12 → 12.1min, mobile phase A are 50% → 95%；12.1 → 15min, mobile phase A are 95%, and flow velocity is 0.3ml/
min；Step 4：Multiple-reaction monitoring is carried out using electron spray positive ion mode, can be from donkey skin, Corium Equi, cattle according to testing result
Donkey skin is distinguished in skin, Corii Caprae seu Oviss and Corii Sus domestica, if five feature peptides are all detected, the skin sample is donkey skin：Donkey source property feature peptide 1 is selected
Select m/z380.7 → 374.82,493.56；Donkey source property feature peptide 2 selects m/z393.24 → 384.42,499.3；Donkey source property feature
Peptide 3 selects m/z421.71 → 585.17,656.26；Donkey source property feature peptide 4 selects m/z400.26 → 500.2,613.27；Donkey source
Property feature peptide 5 select m/z408.26 → 345.18,516.22；MRM scannings are carried out as ion pair；In step 2, the degeneration
Buffer is 0.5MTris-HCl, 2.75mM ethylenediaminetetraacetic acid, 0.5M guanidine hydrochlorides, the buffer of pH8.0；In step 4, institute
Electron spray positive ion mode is stated using triple quadrupole bar mass spectrum as detector, described its electric spray ion source is ESI+；One group of donkey
Source property feature peptide in terms of Colla Corii Asini discriminating in application flow, comprise the steps：Step one：Using trypsin digestion：Claim
Take 0.2g glue class samples and be dissolved in 1% ammonium hydrogencarbonate buffer of 100ml, add 37 DEG C of temperature bath enzymolysis of trypsin；Super filter tube is centrifuged
Filtrate is collected by filtration；Step 2：Carry out LC-MS detection：The chromatographic column for adopting is with octadecylsilane chemically bonded silica as filling
Agent；With 0.1% aqueous formic acid as mobile phase A, with acetonitrile as Mobile phase B, gradient elution：0 → 5min, mobile phase A are 95%；
5 → 8min, mobile phase A are 95% → 92%；8 → 10min, mobile phase A are 92% → 50%；10 → 12min, mobile phase A is
50%；12 → 12.1min, mobile phase A are 50% → 95%；12.1 → 15min, mobile phase A are 95%, and flow velocity is 0.3ml/
min；Step 3：Multiple-reaction monitoring is carried out using electron spray positive ion mode, according to testing result can from Colla Corii Asini, Corium Equi glue,
Colla Corii Asini is distinguished in animal glue, Corii Caprae seu Oviss glue and pig skin gelatinum：If five feature peptide is all detected, the sample is Colla Corii Asini：Donkey source property
Feature peptide 1 selects m/z380.7 → 374.82,493.56；Donkey source property feature peptide 2 selects m/z393.24 → 384.42,499.3；
Donkey source property feature peptide 3 selects m/z421.71 → 585.17,656.26；Donkey source property feature peptide 4 selects m/z400.26 → 500.2,
613.27；Donkey source property feature peptide 5 selects m/z408.26 → 345.18,516.22；MRM scannings are carried out as ion pair；Step 3
In, using triple quadrupole bar mass spectrum as detector, described its electric spray ion source is ESI+ to the electron spray positive ion mode.
The characteristics of the present embodiment is, one group of donkey source property feature peptide, including five donkey source property feature peptides, five donkey sources
Property feature peptide be respectively donkey source property feature peptide 1GPTGEPGKPGDK；Donkey source property feature peptide 2GATGPAGVR；Donkey source property feature peptide
3GEAGPQGAR；Donkey source property feature peptide 4GEIGNPGR；Donkey source property feature peptide 5GEIGNPGR (P hydroxylatings), one group of donkey source property are special
Application flow during peptide is levied in terms of donkey skin discriminating, comprises the steps：Step one：By skin sample unhairing, subcutaneous fat and impurity；
Step 2：Using trypsin digestion；Step 3：Carry out LC-MS detection；Step 4：Entered using electron spray positive ion mode
Row multiple-reaction monitoring, can distinguish donkey skin from donkey skin, Corium Equi, Corii Bovis seu Bubali, Corii Caprae seu Oviss and Corii Sus domestica according to testing result, if five special
Levy peptide all to detect, then the skin sample is donkey skin, one group of donkey source property feature peptide in terms of Colla Corii Asini discriminating in application flow include as
Lower step：Step one：Using trypsin digestion；Step 2：Carry out LC-MS detection；Step 3：Using electron spray just from
Subpattern carries out multiple-reaction monitoring, can be from area in Colla Corii Asini, Corium Equi glue, animal glue, Corii Caprae seu Oviss glue and pig skin gelatinum according to testing result
Divide Colla Corii Asini：If five feature peptides are all detected, the sample is Colla Corii Asini, and the present invention is by Uniprot offers
The result of Peptidemass functional simulation trypsin digestion different plant species collagen protein, and by by the collagen protein sequence of donkey
Row obtain characteristic peptide sequence of the donkey relative to other species with the sequence alignment of other species collagen protein, and are applied
To in the qualitative detection of donkey skin and Colla Corii Asini.
In the present embodiment, the ammonium hydrogen carbonate used in test, trypsin is purchased from Sigma companies, acetonitrile, formic acid purchase
From Merck companies.All reagents are chromatographically pure.One group of donkey source property feature peptide in terms of donkey skin discriminating in application flow include
Following steps：Step one：By skin sample unhairing, subcutaneous fat and impurity；Step 2：Using trypsin digestion：Weigh skin class sample
Product, carry out ungrease treatment using acetone, add ultra-pure water to dissolve skin, weigh 1.5mg and add deionized water to match somebody with somebody after lyophilization
Into certain density protein sample, denaturation buffer is added, denaturation buffer is 0.5MTris-HCl, 2.75mM ethylenediamine tetrem
Acid, 0.5M guanidine hydrochlorides, the buffer of pH8.0, carry out ultrafiltration and are replaced into ammonium hydrogencarbonate buffer, add 37 DEG C of temperature baths of trypsin
Enzymolysis, super filter tube centrifugal filtration collect filtrate；Step 3：Carry out LC-MS detection：The testing conditions of HPLC-TQ-SMS/MS
It is as follows：The chromatographic column for adopting is with octadecylsilane chemically bonded silica as filler；With 0.1% aqueous formic acid as mobile phase A, with
Acetonitrile is Mobile phase B, gradient elution：0 → 5min, mobile phase A are 95%；5 → 8min, mobile phase A are 95% → 92%；8→
10min, mobile phase A are 92% → 50%；10 → 12min, mobile phase A are 50%；12 → 12.1min, mobile phase A be 50% →
95%；12.1 → 15min, mobile phase A are 95%, and flow velocity is 0.3ml/min；Step 4：Entered using electron spray positive ion mode
Row multiple-reaction monitoring, can distinguish donkey skin from donkey skin, Corium Equi, Corii Bovis seu Bubali, Corii Caprae seu Oviss and Corii Sus domestica according to testing result, if five special
Levy peptide all to detect, then the skin sample is donkey skin：Donkey source property feature peptide 1 selects m/z380.7 → 374.82,493.56；Donkey source property is special
Levy peptide 2 and select m/z393.24 → 384.42,499.3；Donkey source property feature peptide 3 selects m/z421.71 → 585.17,656.26；Donkey
Source property feature peptide 4 selects m/z400.26 → 500.2,613.27；Donkey source property feature peptide 5 selects m/z408.26 → 345.18,
516.22；MRM scannings are carried out as ion pair.
In the present embodiment, using ThermoScientific companies Fusion-Orbitrap high-resolution mass spectrometers, institute
State its ion source spray source is received for Nanospray Flex.Collagen protein is the major protein in animal dermis, therefore the present invention exists
Suitable polypeptide is selected in collagen protein as the characteristic polypeptide of donkey skin collagens albumen.There is provided using Uniport
PeptideMass functional simulations obtain trypsin digestion donkey skin, Corium Equi, Corii Bovis seu Bubali, Corii Caprae seu Oviss, the result of pigskin collagen；Profit
The sequence of donkey skin, Corium Equi, Corii Bovis seu Bubali, Corii Caprae seu Oviss, pigskin collagen is compared with molecular biology software MEGA5.0.In order to
Checking donkey skin feature peptide, have selected donkey skin, Corium Equi, Corii Bovis seu Bubali, Corii Caprae seu Oviss, Corii Sus domestica, according to sample pretreating method mentioned above with
And EASY-nLC1000-Orbitrap Fusion LC-MS analysis methods, the molecular weight and two of testing result display feature peptide
Level mass spectrum is all consistent with theoretical value.
In the present embodiment, the application flow during one group of donkey source property feature peptide is in terms of Colla Corii Asini discriminating, including following step
Suddenly：Step one：Using trypsin digestion：Weigh 0.2g glue class samples and be dissolved in 1% ammonium hydrogencarbonate buffer of 100ml, add pancreas
7 DEG C of temperature bath enzymolysis of protease 3；Filtrate is collected in super filter tube centrifugal filtration；Step 2：Carry out LC-MS detection：HPLC-TQ-S
The condition of MS/MS is as follows：The chromatographic column for adopting (chromatograph column internal diameter 2.1mm) with octadecylsilane chemically bonded silica as filler；
With 0.1% aqueous formic acid as mobile phase A, with acetonitrile as Mobile phase B, gradient elution：0 → 5min, mobile phase A are 95%；5→
8min, mobile phase A are 95% → 92%；8 → 10min, mobile phase A are 92% → 50%；10 → 12min, mobile phase A is
50%；12 → 12.1min, mobile phase A are 50% → 95%；12.1 → 15min, mobile phase A are 95%, and flow velocity is 0.3ml/
min；Step 3：Multiple-reaction monitoring is carried out using electron spray positive ion mode, electron spray positive ion mode is with triple quadrupole bar matter
Used as detector, its electric spray ion source is ESI+ to spectrum, can be from Colla Corii Asini, Corium Equi glue, animal glue, Corii Caprae seu Oviss glue according to testing result
With Colla Corii Asini is distinguished in pig skin gelatinum：If five feature peptide is all detected, the sample is Colla Corii Asini：Donkey source property feature peptide 1 selects m/
Z380.7 → 374.82,493.56, as shown in figure 3, A：Extract ion stream；B：First mass spectrometric；C：Second order mses figure；；Donkey source property
The selection of feature peptide 2 m/z393.24 → 384.42,499.3, as shown in figure 4, A：Extract ion stream；B：First mass spectrometric；C：Two grades of matter
Spectrogram；Donkey source property feature peptide 3 selects m/z421.71 → 585.17, and 656.26, as shown in figure 5, A：Extract ion stream；B：One-level
Mass spectrum；C：Second order mses figure；Donkey source property feature peptide 4 selects m/z400.26 → 500.2, and 613.27, as shown in fig. 6, A：Extract from
Subflow；B：First mass spectrometric；C：Second order mses figure；Donkey source property feature peptide 5 selects m/z408.26 → 345.18,516.22, such as Fig. 7
It is shown, A：Extract ion stream；B：First mass spectrometric；C：Second order mses figure；MRM scannings are carried out as ion pair.
In the present embodiment, embodiment 1, the identification of one group of donkey source property feature peptide:
Collagen protein is the major protein in animal dermis, therefore the present invention selects suitable polypeptide to make in collagen protein
For the characteristic polypeptide of donkey skin collagens albumen.Trypsin enzyme is obtained using the PeptideMass functional simulations that Uniport is provided
Solution donkey skin, Corium Equi, Corii Bovis seu Bubali, Corii Caprae seu Oviss, the result of pigskin collagen；Using molecular biology software MEGA5.0 to donkey skin, horse
Skin, Corii Bovis seu Bubali, Corii Caprae seu Oviss, the sequence of pigskin collagen are compared, so as to the feature peptide sequence of the theory of acquisition.In order to verify donkey
Skin feature peptide, have selected donkey skin, Corium Equi, Corii Bovis seu Bubali, Corii Caprae seu Oviss, Corii Sus domestica, according to sample pretreating method mentioned above and EASY-
NLC 1000-Orbitrap Fusion LC-MS analysis methods.Testing result as shown in fig. 3 to 7, the molecular weight of feature peptide and
Second order mses are all consistent with theoretical value.
Wherein, the testing conditions of EASY-nLC 1000-Orbitrap Fusion LC-MS analysis methods are：First,
Separated using 1000 nanoliters of liquid phase systems of EASY-nLC.Wherein using the formic acid water of 0.1% that mobile phase A is 2% acetonitrile
Solution and Mobile phase B are the aqueous formic acid of the 0.1% of 98% acetonitrile as detectable.Wherein staff can carry out equipment
Arrange, gradient is set to 0 → 15min of first stage, and flow velocity is 300nLmin-1, and mobile phase A is 100%, second-order
15 → 70min of section, flow velocity are 300nLmin-1, and mobile phase A is 93% → 78%, 70 → 90min of phase III, and flow velocity is
300nLmin-1, mobile phase A are 78% → 65%, 90 → 95min of fourth stage, and flow velocity is 300nLmin-1, mobile phase A
For 65% → 10%, the 5th 95 → 105min of stage, flow velocity are 450nLmin-1, and mobile phase A is 10%, sample size therein
It is 1 μ l.Second, taken off using the ReproSil-Pur C18-AQ Trap column of 0.2mm × 3.5cm (5 μm of particle diameters)
Salt enrichment work, wherein the instrument for adopting makes the ReproSil-Pur C18-AQ of 75 μ m 25cm (3 μm of particle diameters) by oneself for laboratory
Nanoliter analytical column separation equipment.3rd, using Thermo Scientific companies Fusion-Orbitrap high-resolution mass spectrometers
Detected, wherein ion source receives spray source for Nanospray Flex.It is analyzed using positive ion mode, spray voltage is
2.1kV, ion transfer capillary temperature are 275 DEG C, and S-Lens efficiencies of transmission are set to 60%.First mass spectrometric adopts Orbitrap
Used as mass analyzer, resolution is 60,000 (m/z=400), and acquisition range is 350-1,650Th.Second order mses using from
Sub- trap is scanned using Rapid Scan patterns as mass analyzer, carries out parent ion using Top20 data dependences pattern
Select, fragmentation is carried out using CID patterns, fragmentation energies NCE are set to 35%, so as to complete to detect work.
In the present embodiment, embodiment 2, the MRM testing conditions of donkey source property feature peptide:
The optimization of MRM parameters is carried out to features above peptide using triple level Four bar LC-MS methods, the MRM ginsengs after optimization
Number is as shown in table 1：
The MRM testing conditions of 1 donkey skin feature peptide of table
| Composition | Parent ion (m/z) | Daughter ion (m/z) | Taper hole voltage (v) | Collision energy (ev) |
| Donkey source property feature peptide 1 | 380.7 | 374.82 | 20 | 10 |
| Donkey source property feature peptide 1 | 380.7 | 493.56 | 20 | 10 |
| Donkey source property feature peptide 2 | 393.24 | 384.42 | 15 | 10 |
| Donkey source property feature peptide 2 | 393.24 | 499.3 | 15 | 16 |
| Donkey source property feature peptide 3 | 421.71 | 585.17 | 18 | 16 |
| Donkey source property feature peptide 3 | 421.71 | 656.26 | 18 | 16 |
| Donkey source property feature peptide 4 | 400.26 | 500.2 | 16 | 15 |
| Donkey source property feature peptide 4 | 400.26 | 613.27 | 16 | 15 |
| Donkey source property feature peptide 5 | 408.26 | 345.18 | 18 | 15 |
| Donkey source property feature peptide 5 | 408.26 | 516.22 | 18 | 15 |
Wherein, triple level Four bar LC-MS methods are with the HPLC-TQ-SMS/MS LC-MS described in claims
Detection method.
Above-mentioned technical proposal only embodies the optimal technical scheme of technical solution of the present invention, those skilled in the art
Some of which part may be made some variation embody the present invention principle, belong to protection scope of the present invention it
It is interior.
Claims (7)
1. one group of donkey source property feature peptide, it is characterised in that including five donkey source property feature peptides, five donkey sources property feature peptide point
Wei not donkey source property feature peptide 1GPTGEPGKPGDK；Donkey source property feature peptide 2GATGPAGVR；Donkey source property feature peptide 3GEAGPQGAR；
Donkey source property feature peptide 4GEIGNPGR；Donkey source property feature peptide 5GEIGNPGR (P hydroxylatings).
2. one group of donkey source property feature peptide according to claim 1, it is characterised in that the donkey source property feature peptide 1 is donkey phase
Peculiar for horse, cattle, sheep, pig institute, donkey source property feature peptide 2 is that donkey is peculiar relative to horse and pig institute, donkey source property feature peptide 3 and donkey source
Property 4 to be donkey peculiar relative to cattle, sheep, pig institute, donkey source property feature peptide 5 is that donkey is peculiar relative to cattle, sheep institute.
3. the application stream during a kind of one group of donkey source property feature peptide according to claim 1-2 is arbitrary is in terms of donkey skin discriminating
Journey, it is characterised in that comprise the steps：
Step one：By skin sample unhairing, subcutaneous fat and impurity；
Step 2：Using trypsin digestion：Skin class sample is weighed, ungrease treatment is carried out using acetone, add ultra-pure water by skin
Dissolving, weighs 1.5mg and adds deionized water to be made into certain density protein sample, add denaturation buffer, enter after lyophilization
Row ultrafiltration is replaced into ammonium hydrogencarbonate buffer, adds 37 DEG C of temperature bath enzymolysis of trypsin, super filter tube centrifugal filtration to collect filtrate；
Step 3：Carry out LC-MS detection：The chromatographic column for adopting is with octadecylsilane chemically bonded silica as filler；With
0.1% aqueous formic acid is mobile phase A, with acetonitrile as Mobile phase B, gradient elution：0 → 5min, mobile phase A are 95%；5→
8min, mobile phase A are 95% → 92%；8 → 10min, mobile phase A are 92% → 50%；10 → 12min, mobile phase A is
50%；12 → 12.1min, mobile phase A are 50% → 95%；12.1 → 15min, mobile phase A are 95%, and flow velocity is 0.3ml/
min；
Step 4：Multiple-reaction monitoring is carried out using electron spray positive ion mode, can be from donkey skin, Corium Equi, cattle according to testing result
Donkey skin is distinguished in skin, Corii Caprae seu Oviss and Corii Sus domestica, if five feature peptides are all detected, the skin sample is donkey skin：Donkey source property feature peptide 1 is selected
Select m/z380.7 → 374.82,493.56；Donkey source property feature peptide 2 selects m/z393.24 → 384.42,499.3；Donkey source property feature
Peptide 3 selects m/z421.71 → 585.17,656.26；Donkey source property feature peptide 4 selects m/z400.26 → 500.2,613.27；Donkey source
Property feature peptide 5 select m/z408.26 → 345.18,516.22；MRM scannings are carried out as ion pair.
4. the application flow during one group of donkey source according to claim 3 property feature peptide is in terms of donkey skin discriminating, its feature exist
In, in step 2, the denaturation buffer be 0.5MTris-HCl, 2.75mM ethylenediaminetetraacetic acid, 0.5M guanidine hydrochlorides, pH8.0
Buffer.
5. the application flow during one group of donkey source according to claim 3 property feature peptide is in terms of donkey skin discriminating, its feature exist
In, in step 4, the electron spray positive ion mode using triple quadrupole bar mass spectrum as detector, described its electric spray ion source
For ESI+.
6. the application stream during a kind of one group of donkey source property feature peptide according to claim 1-2 is arbitrary is in terms of Colla Corii Asini discriminating
Journey, it is characterised in that comprise the steps：
Step one：Using trypsin digestion：Weigh 0.2g glue class samples and be dissolved in 1% ammonium hydrogencarbonate buffer of 100ml, add
37 DEG C of temperature bath enzymolysis of trypsin；Filtrate is collected in super filter tube centrifugal filtration；
Step 2：Carry out LC-MS detection：The chromatographic column for adopting is with octadecylsilane chemically bonded silica as filler；With
0.1% aqueous formic acid is mobile phase A, with acetonitrile as Mobile phase B, gradient elution：0 → 5min, mobile phase A are 95%；5→
8min, mobile phase A are 95% → 92%；8 → 10min, mobile phase A are 92% → 50%；10 → 12min, mobile phase A is
50%；12 → 12.1min, mobile phase A are 50% → 95%；12.1 → 15min, mobile phase A are 95%, and flow velocity is 0.3ml/
min；
Step 3：Multiple-reaction monitoring is carried out using electron spray positive ion mode, according to testing result can from Colla Corii Asini, Corium Equi glue,
Colla Corii Asini is distinguished in animal glue, Corii Caprae seu Oviss glue and pig skin gelatinum：If five feature peptide is all detected, the sample is Colla Corii Asini：Donkey source property
Feature peptide 1 selects m/z380.7 → 374.82,493.56；Donkey source property feature peptide 2 selects m/z393.24 → 384.42,499.3；
Donkey source property feature peptide 3 selects m/z421.71 → 585.17,656.26；Donkey source property feature peptide 4 selects m/z400.26 → 500.2,
613.27；Donkey source property feature peptide 5 selects m/z408.26 → 345.18,516.22；MRM scannings are carried out as ion pair.
7. the application flow during one group of donkey source according to claim 6 property feature peptide is in terms of Colla Corii Asini discriminating, its feature exist
In, in step 3, the electron spray positive ion mode using triple quadrupole bar mass spectrum as detector, described its electric spray ion source
For ESI+.
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| CN111855863A (en) * | 2020-08-14 | 2020-10-30 | 江西师范大学 | Method for identifying gelatin source in donkey-hide gelatin product |
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| CN111855863A (en) * | 2020-08-14 | 2020-10-30 | 江西师范大学 | Method for identifying gelatin source in donkey-hide gelatin product |
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| 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 |
| CN115015409A (en) * | 2022-05-23 | 2022-09-06 | 山东省食品药品检验研究院 | Method for establishing LCMS (liquid Crystal display System) characteristic spectrum of donkey-hide gelatin polypeptide and quality evaluation method thereof |
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