CN113173971B - Characteristic peptide segment for identifying formula granules containing deer horns or deer skins and detection method thereof - Google Patents
Characteristic peptide segment for identifying formula granules containing deer horns or deer skins and detection method thereof Download PDFInfo
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Abstract
The invention discloses a characteristic peptide segment for identifying formula particles containing deer horns or deer skin and a detection method thereof, wherein the characteristic peptide segment is peptide 1-peptide 4, such as a sequence table 1, the invention uses peptide 4 as reference, and uses peptide 1 peak area A Peptide 1 Area A with peptide 4 Peak Peptide 4 Or peptide 2 peak area A Peptide 2 Area A with peptide 4 Peak Peptide 4 Or peptide 3 peak area A Peptide 3 Area A with peptide 4 Peak Peptide 4 The ratio of (A) to (B) is used for quality control of the deer horn or deer skin formula granules. The invention adopts liquid chromatography-tandem mass spectrometry to carry out multi-reaction detection in an electrospray positive ion mode, wherein the peptide 1 parent ion is 612.2, and the daughter ions are 747.4 and 464.2; peptide 2 parent ion 871.4 and daughter ions 726.4, 862.9; the peptide 3 parent ion is 808.9, the daughter ion is 892.4 and 554.3, the peptide 4 parent ion is 596.8, and the daughter ion 518.7 is the deer-horn glue characteristic peptide, so that the deer-horn glue and the deer-horn glue can be identified and distinguished.
Description
Technical Field
The invention relates to a deer-derived characteristic peptide segment and a detection method thereof, in particular to the quality control of identifying deer-horn glue and deer-hide glue, as well as deer-horn glue formula particles, deer-horn formula particles, and medicaments, health care products and foods which take the deer-horn glue as raw materials.
Technical Field
The gelatin medicinal materials comprise colla Corii Asini, colla Cornus Cervi, gelatinum oxhide, etc., more than 80% of the components are collagen of different classes, including type I collagen alpha 1 chain (COL1A1), type I collagen alpha 2 chain (COL1A2), type II collagen alpha 1 chain (COL2A1), type III collagen alpha 1 chain (COL3A1), etc., wherein the peptide segment from COL1A2 is mainly used. COL1A2 is a highly conserved protein, widely present in different animal species, and is one of the important protein components constituting gelatin-type medicinal materials.
The deer-horn glue and the deer-skin glue are both from sika deer or red deer, the two are rare glue Chinese medicinal materials, the deer-horn glue is solid glue prepared by decocting and concentrating sika deer or red deer horn, and the deer-skin glue is solid glue prepared by decocting and concentrating dried skin or fresh skin of sika deer or red deer. The deer horn is the ossified horn of red deer or sika deer, the price is far higher than deer skin, and the phenomenon that deer skin glue is mixed with deer horn glue to be secondary good exists in the market. How to identify the deerhorn glue and the deerhorn glue is a difficult problem of identification research of glue medicinal materials, and challenges are brought to the identification of the deerhorn glue and the identification of counterfeit products of the deerhorn glue mixed with the deerhorn glue.
The antler glue formula particles and the antler glue formula particles are prepared by carrying out complex processing on medicines, health-care products and foods which take the antler glue as a raw material, if the deerhorn glue is adulterated, the deerhorn glue and the deerhorn skin are difficult to distinguish from each other only in appearance, the protein compositions of the antler and the deerhorn skin are the same, and the deerhorn glue and the deerhorn skin glue cannot be distinguished by the existing species specific peptide.
The invention content is as follows:
the invention aims to: the invention determines the ratio of the relative contents of 4 deer-derived peptide fragments through a large number of experimental screens, thereby being used for identifying the deer-horn glue and the deer-skin glue. The method has the advantages of high specificity, high sensitivity, and simple operation, and can be used for quality control of colla Cornus Cervi and its related products.
In order to achieve the purpose, the invention adopts the following technical scheme:
a characteristic peptide fragment for identifying formula granules containing deer horns or deer hides, characterized in that the characteristic peptide is:
peptide 1:
Gly-Phe-Hyp-Gly-Thr-Pro-Gly-Leu-Hyp-Gly-Phe-Lys
peptide 2:
Gly-Pro-Ile-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Asp-Lys-Gly-Glu-Thr-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Pro-Thr-Gly-Ala-Arg
peptide 3:
Gly-Glu-Leu-Gly-Pro-Val-Gly-Asn-Hyp-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Arg
peptide 4:
Gly-Val-Hyp-Gly-Pro-Hyp-Gly-Ala-Val-Gly-Pro-Ala-Gly-Lys。
an assay for identifying characteristic peptide fragments of a granulate formulation containing deer horns or deer skins, comprising the steps of:
(1) preparing the peptide 1- peptide 4, 4 deer-derived characteristic peptides into a mixed reference solution;
(2) after the deer-horn glue formula particles to be detected and the deer-horn glue samples are subjected to enzyme digestion by trypsin, the enzymolysis liquid and the mixed reference substance solution of the peptides 1-4 in the step (1) are injected into a liquid chromatograph-mass spectrometer, the reference substance solution is controlled by deer-derived characteristic peptides, an ESI positive ion mode and a multi-reaction monitoring mode are adopted, and the selected ion pairs comprise: peptide 1: m/z 612.2 (double charge) → 747.4, peptide 2: m/z 871.4 (three charges) → 726.4, peptide 3: m/z 808.9 (double charge) → 892.4, peptide 4: m/z 596.8 (double charge) → 518.7; wherein the peptide 4 is taken as a reference, and the peak area A of the peptide 1 is taken as Peptide 1 Area A with peptide 4 Peak Peptide 4 Or peptide 2 peak area A Peptide 2 Area A with peptide 4 Peak Peptide 4 Or peptide 3 peak area A Peptide 3 Area A with peptide 4 Peak Peptide 4 The ratio of (A) to (B) determines whether the sample source is deer-horn glue or deer-skin glue.
Preferably, the enzyme digestion method comprises the following steps: taking 10mg of a formula particle or glue medicinal material sample to be detected, adding 5ml of phosphate buffer solution, performing ultrasonic treatment to completely dissolve the sample, centrifuging at 12000rpm for 20min, taking 150 mu l of supernatant, placing the supernatant in a 2ml centrifuge tube, diluting with 1ml of 50mM PBS, adding a proper amount of trypsin, shaking up, performing full enzymolysis, adding 60 mu l of 10% trifluoroacetic acid solution to terminate the reaction, and centrifuging at 12000rpm for 20min to obtain glue medicinal material enzymolysis solution, and placing the glue medicinal material enzymolysis solution at-20 ℃ for storage for later use.
Preferably, the detection method for identifying the characteristic peptide segment of the formula particle containing the deer horn or the deer skin is characterized in that the mass concentration of the trypsin is 0.1-10%.
Preferably, the detection method for identifying the characteristic peptide segment of the formula particle containing the deer horn or the deer skin comprises the following steps of: constant temperature enzymolysis at 37 ℃, microwave-assisted enzymolysis, ultrasonic-assisted enzymolysis and enzyme immobilization enzymolysis.
Preferably, the detection method for identifying the characteristic peptide segment of the formula particle containing deer horn or deer skin comprises the following liquid phase conditions detected by a liquid chromatograph-mass spectrometer: the chromatographic column is 1.7 mu m Waters C 18 A column with a specification of 2.1 μm × 100mm, a sample loading amount of 2 μ l, a flow rate of 0.3ml/min, 0-3.5 min, 10-30% A linear gradient elution, 3.5-4 min, 30-10% A linear gradient elution, 4-6 min, 10% A elution.
The mass spectrum conditions detected by the LC-MS are as follows: electrospray positive ion mode ESI +, mass spectrometry parameters were: the ion source temperature is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60 psi; ion source gas 2, 60 psi;
setting the ion pair conditions corresponding to the characteristic peptides as follows:
peptide 1: m/z 612.2 (double charge) → 747.4, DP 128.74, CE 31.66;
peptide 2: m/z 871.4 (triply) → 726.4, DP 131.57, CE 34.95;
peptide 3: m/z 808.9 (double charge) → 892.4, DP 141.54, CE 31.07;
peptide 4: m/z 596.8 (double charge) → 518.7, DP 145.69, CE 35.54.
Preferably, the detection method for identifying the characteristic peptide segment of the formula particle containing deer horn or deer skin,
antler glue formula particle A Peptide 1 /A Peptide 4 Mean value ofIs 7.81 +/-0.73, A Peptide 2 /A Peptide 4 Mean value of 2.51. + -. 0.53, A Peptide 3 /A Peptide 4 The average value is 3.48 +/-0.71;
deer skin glue A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 The values of (A) are shown in Table 3 Peptide 1 /A Peptide 4 Mean value of 0.37. + -. 0.09, A Peptide 2 /A Peptide 4 Average value of 0.14. + -. 0.02, A Peptide 3 /A Peptide 4 The average value was 0.20. + -. 0.04.
Preferably, the formula particle containing the antler or the deer skin comprises a antler glue formula particle, a antler formula particle, a medicine, a health product, a food and the like which take the antler glue as a raw material.
Has the advantages that: compared with the prior art, the invention has the following advantages:
according to the invention, through a large number of experimental screening, researches show that the ratio of the relative content of 3 deer-derived peptide fragments of peptides 1-3 and peptides 4 has specificity, so that the deer-horn glue and the deer-skin glue can be sequentially identified. The method has high specificity, high sensitivity, and simple operation, and can be used for quality control of colla Cornus Cervi. Thereby overcoming the defects that the deer-horn glue and the deer-skin glue are difficult to identify from the appearance and the specific peptide segment is difficult to identify in the prior art, and obtaining good technical progress.
Drawings
FIG. 1 is a mass spectrum of peptide 1.
FIG. 2 is a mass spectrum of peptide 2.
FIG. 3 is a mass spectrum of peptide 3.
FIG. 4 is a mass spectrum of peptide 4.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The invention is further illustrated below with reference to specific examples, which should not be construed as limiting the invention.
Example 1
A deer-derived characteristic peptide has 4 characteristic peptide sequences as shown in a sequence table 1:
peptide 1:
Gly-Phe-Hyp-Gly-Thr-Pro-Gly-Leu-Hyp-Gly-Phe-Lys
peptide 2:
Gly-Pro-Ile-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Asp-Lys-Gly-Glu-Thr-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Pro-Thr-Gly-Ala-Arg
peptide 3:
Gly-Glu-Leu-Gly-Pro-Val-Gly-Asn-Hyp-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Arg
peptide 4:
Gly-Val-Hyp-Gly-Pro-Hyp-Gly-Ala-Val-Gly-Pro-Ala-Gly-Lys。
example 2 antler glue A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 Measurement of (2)
Taking 5 batches of antler glue samples, taking 10mg of antler glue samples in each batch, adding 5ml of phosphate buffer solution, performing ultrasonic treatment to completely dissolve the samples, centrifuging at 12000rpm for 20min, taking 150 mu l of supernatant, placing the supernatant in a 2ml centrifuge tube, diluting with 1ml of 50mM PBS, adding 1% trypsin (w/v), shaking up, performing enzymolysis at constant temperature of 37 ℃ for 12h, adding 60 mu l of 10% TFA solution after enzymolysis to terminate the reaction, and centrifuging at 12000rpm for 20min to obtain antler glue enzymolysis liquid, and storing at-20 ℃ for later use.
Injecting the 5 batches of antler glue enzymatic hydrolysate into a liquid chromatograph-mass spectrometer with the sample volume of 1 mu g, wherein the liquid phase conditions detected by the liquid chromatograph-mass spectrometer are as follows: the chromatographic column is a 1.7 mu m C18 reversed phase chromatographic column (2.1 mu m multiplied by 100mm), the flow rate is 0.3ml/min, the mobile phase A (acetonitrile), the mobile phase B (0.1% formic acid), 0-3.5 min, 10-30% A linear gradient elution, 3.5-4 min, 30-10% A linear gradient elution, 4-6 min and 10% A elution. The mass spectrum conditions detected by the LC-MS are as follows: electrospray positive ion mode was (ESI +), mass spectrometry parameters were: the ion source temperature is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60 psi; the ion source gas was 2, 60 psi. Setting the ion pair conditions corresponding to the characteristic peptides as follows:
peptide 1: m/z 612.2 (double charge) → 747.4, DP 128.74, CE 31.66;
peptide 2: m/z 871.4 (triply) → 726.4, DP 131.57, CE 34.95;
peptide 3: m/z 808.9 (double charge) → 892.4, DP 141.54, CE 31.07;
peptide 4: m/z 596.8 (double charge) → 518.7, DP 145.69, CE 35.54.
5 batches of antler glue A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 The values of (A) are shown in Table 1 Peptide 1 /A Peptide 4 Mean value 7.12. + -. 0.55, A Peptide 2 /A Peptide 4 Mean value of 2.48. + -. 0.33, A Peptide 3 /A Peptide 4 The average was 3.44. + -. 0.51.
Example 3 Degelating formulations A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 Measurement of (2)
Taking 5 batches of antler glue formula particle samples, taking 20mg of antler glue formula particle samples in each batch, adding 5ml of phosphate buffer solution, performing ultrasonic treatment to completely dissolve the samples, centrifuging at 12000rpm for 20min, taking 150 mu l of supernatant, placing the supernatant in a 2ml centrifuge tube, diluting with 1ml of 50mM PBS, adding 1% trypsin (w/v), shaking up, performing enzymolysis at constant temperature of 37 ℃ for 12h, adding 60 mu l of 10% TFA solution after enzymolysis to terminate the reaction, and centrifuging at 12000rpm for 20min to obtain antler glue formula particle enzymolysis liquid, and storing at-20 ℃ for later use.
Injecting the 5 batches of the antler glue formula particle enzymolysis liquid into a liquid chromatograph-mass spectrometer, wherein the sample injection amount is 1 mu g, and the liquid phase conditions detected by the liquid chromatograph-mass spectrometer are as follows: the chromatographic column is a 1.7 mu m C18 reversed phase chromatographic column (2.1 mu m multiplied by 100mm), the flow rate is 0.3ml/min, the mobile phase A (acetonitrile), the mobile phase B (0.1% formic acid), 0-3.5 min, 10-30% A linear gradient elution, 3.5-4 min, 30-10% A linear gradient elution, 4-6 min and 10% A elution. The mass spectrum conditions detected by the LC-MS are as follows: electrospray positive ion mode (ESI +), mass spectral parameters: the ion source temperature is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60 psi; the ion source gas was 2, 60 psi. Setting the ion pair conditions corresponding to the characteristic peptides as follows:
peptide 1: m/z 612.2 (double charge) → 747.4, DP 128.74, CE 31.66;
peptide 2: m/z 871.4 (triply) → 726.4, DP 131.57, CE 34.95;
peptide 3: m/z 808.9 (double charge) → 892.4, DP 141.54, CE 31.07;
peptide 4: m/z 596.8 (double charge) → 518.7, DP 145.69, CE 35.54.
5 batches of antler glue formula granules A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 The values of (A) are shown in Table 2 Peptide 1 /A Peptide 4 Mean value of 7.81. + -. 0.73, A Peptide 2 /A Peptide 4 Mean value of 2.51. + -. 0.53, A Peptide 3 /A Peptide 4 The mean value was 3.48. + -. 0.71.
Example 4 deer skin glue A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 Measurement of (2)
Taking 5 batches of deer skin glue samples, taking 20mg of each batch of deer skin glue samples, adding 5ml of phosphate buffer solution, performing ultrasonic treatment to completely dissolve the samples, centrifuging at 12000rpm for 20min, taking 150 mu l of supernatant, placing the supernatant into a 2ml centrifuge tube, diluting with 1ml of 50mM PBS, adding 1% trypsin (w/v), shaking up, performing enzymolysis at the constant temperature of 37 ℃ for 12h, adding 60 mu l of 10% TFA solution after enzymolysis to terminate the reaction, and centrifuging at 12000rpm for 20min to obtain deer skin glue enzymolysis liquid, and storing at-20 ℃ for later use.
Injecting the 5 batches of the deer skin glue enzymatic hydrolysate into a LC-MS, wherein the sample volume is 1 mu g, and the LC conditions detected by the LC-MS are as follows: the chromatographic column is a 1.7 mu m C18 reversed phase chromatographic column (2.1 mu m multiplied by 100mm), the flow rate is 0.3ml/min, the mobile phase A (acetonitrile), the mobile phase B (0.1% formic acid), 0-3.5 min, 10-30% A linear gradient elution, 3.5-4 min, 30-10% A linear gradient elution, 4-6 min and 10% A elution. The mass spectrum conditions detected by the LC-MS are as follows: electrospray positive ion mode (ESI +), mass spectrometry parameters were: the ion source temperature is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60 psi; the ion source gas was 2, 60 psi. Setting the ion pair conditions corresponding to the characteristic peptides as follows:
peptide 1: m/z 612.2 (double charge) → 747.4, DP 128.74, CE 31.66;
peptide 2: m/z 871.4 (triply) → 726.4, DP 131.57, CE 34.95;
peptide 3: m/z 808.9 (double charge) → 892.4, DP 141.54, CE 31.07;
peptide 4: m/z 596.8 (double charge) → 518.7, DP 145.69, CE 35.54.
5 batches of deerskin glue A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 The values are shown in Table 3, A Peptide 1 /A Peptide 4 Mean value of 0.37. + -. 0.09, A Peptide 2 /A Peptide 4 Mean value of 0.14. + -. 0.02, A Peptide 3 /A Peptide 4 The average value was 0.20. + -. 0.04.
TABLE 1 deer-horn Gum A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 As a result of (A)
TABLE 2 Deerhorn Gum formulation granule A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 As a result of (A)
TABLE 3 deer skin glue A Peptide 1 /A Peptide 4 、A Peptide 2 /A Peptide 4 、A Peptide 3 /A Peptide 4 As a result of (A)
Sequence listing
<110> Jiangyin Tianjiang pharmaceutical Co., Ltd
Nanjing University of Chinese Medicine
<120> characteristic peptide fragment for identifying formula granule containing deer horn or deer skin and detection method thereof
<141> 2021-04-20
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 1
Gly Phe Pro Gly Thr Pro Gly Leu Pro Gly Phe Lys
1 5 10
<210> 2
<211> 30
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 2
Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly
1 5 10 15
Glu Thr Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg
20 25 30
<210> 3
<211> 18
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 3
Gly Glu Leu Gly Pro Val Gly Asn Pro Gly Pro Ala Gly Pro Ala Gly
1 5 10 15
Pro Arg
<210> 4
<211> 14
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 4
Gly Val Pro Gly Pro Pro Gly Ala Val Gly Pro Ala Gly Lys
1 5 10
Claims (5)
1. A detection method for identifying deer-horn glue or deer-skin glue is characterized by comprising the following steps:
(1) preparing 4 deer-derived characteristic peptide fragments into a mixed reference solution; the characteristic peptide segment is as follows:
peptide 1:
Gly-Phe-Hyp-Gly-Thr-Pro-Gly-Leu-Hyp-Gly-Phe-Lys
peptide 2:
Gly-Pro-Ile-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Asp-Lys-Gly-Glu-Thr-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Pro-Thr-Gly-Ala-Arg
peptide 3:
Gly-Glu-Leu-Gly-Pro-Val-Gly-Asn-Hyp-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Arg
peptide 4:
Gly-Val-Hyp-Gly-Pro-Hyp-Gly-Ala-Val-Gly-Pro-Ala-Gly-Lys；
(2) after the deer-horn glue formula particles to be detected and the deer-horn glue samples are subjected to enzyme digestion by trypsin, the enzymolysis liquid and the mixed reference substance solution of the peptides 1-4 in the step (1) are injected into a liquid chromatograph-mass spectrometer, the reference substance solution is compared by deer-derived characteristic peptide fragments, an ESI positive ion mode and a multi-reaction monitoring mode are adopted, and the selected ion pairs comprise: peptide 1: m/z 612.2 double charge → 747.4, peptide 2: m/z 871.4 three charges → 726.4 for detection, peptide 3: m/z 808.9 double charge → 892.4, peptide 4: m/z 596.8 double charge → 518.7 for detection; wherein the peptide 4 is taken as a reference, and the peak area A of the peptide 1 is taken as Peptide 1 Area A with peptide 4 Peak Peptide 4 Or peptide 2 peak area A Peptide 2 Area A with peptide 4 Peak Peptide 4 Or peptide 3 peak area A Peptide 3 Area A with peptide 4 Peak Peptide 4 Determining whether the sample source is deer horn glue or deer skin glue;
antler glue formula particle A Peptide 1 /A Peptide 4 Mean value 7.81. + -. 0.73, A Peptide 2 /A Peptide 4 Mean value of 2.51. + -. 0.53, A Peptide 3 /A Peptide 4 The average value is 3.48 +/-0.71;
deer skin glue A Peptide 1 /A Peptide 4 Mean value of 0.37. + -. 0.09, A Peptide 2 /A Peptide 4 Average value of 0.14. + -. 0.02, A Peptide 3 /A Peptide 4 The average value was 0.20. + -. 0.04.
2. The detection method for identifying deer-horn glue or deer-hide glue according to claim 1, wherein the enzyme digestion method comprises: taking 10mg of the deer-horn glue formula particles or deer-skin glue samples to be detected, adding 5ml of phosphate buffer solution, carrying out ultrasonic treatment to completely dissolve the samples, centrifuging at 12000rpm for 20min, taking 150 mu l of supernatant, placing the supernatant into a 2ml centrifuge tube, diluting with 1ml of 50mM PBS, adding a proper amount of trypsin, shaking up, carrying out full enzymolysis, adding 60 mu l of 10% trifluoroacetic acid solution to terminate the reaction, centrifuging at 12000rpm for 20min to obtain glue medicinal material enzymolysis liquid, and placing the glue medicinal material enzymolysis liquid at-20 ℃ for storage for later use.
3. The detection method for identifying deer-horn glue or deer-hide glue according to claim 2, wherein the mass concentration of trypsin is 0.1-10%.
4. The detection method for identifying deer-horn glue or deer-hide glue according to claim 2, wherein the enzymatic hydrolysis method comprises: constant temperature enzymolysis at 37 ℃, microwave-assisted enzymolysis, ultrasonic-assisted enzymolysis or enzyme-immobilized enzymolysis.
5. The detection method for identifying deer-horn glue or deer-hide glue according to claim 2, wherein the liquid phase conditions detected by the LC-MS are as follows: the chromatographic column is 1.7 mu m Waters C 18 A column with a specification of 2.1 μm × 100mm, a sample loading amount of 2 μ l, a flow rate of 0.3ml/min, 0-3.5 min, 10-30% A linear gradient elution, 3.5-4 min, 30-10% A linear gradient elution, 4-6 min, 10% A elution;
the mass spectrum conditions detected by the LC-MS are as follows: electrospray positive ion mode ESI +, mass spectrometry parameters were: the ion source temperature is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60 psi; ion source gas 2, 60 psi;
setting the ion pair conditions corresponding to the characteristic peptides as follows:
peptide 1: m/z 612.2 double charge → 747.4, DP =128.74, CE = 31.66;
peptide 2: m/z 871.4 three charges → 726.4, DP =131.57, CE = 34.95;
peptide 3: m/z 808.9 double charge → 892.4, DP =141.54, CE = 31.07;
peptide 4: m/z 596.8 double charge → 518.7, DP =145.69, CE = 35.54.
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| CN112098579A (en) * | 2020-09-01 | 2020-12-18 | 南京中医药大学 | Deer-derived characteristic peptide fragment and detection method thereof |
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