CN115572322A - Elk horn characteristic peptide segment and detection method thereof - Google Patents
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Abstract
The invention discloses an elk characteristic peptide segment and a detection method thereof, the invention screens out elk characteristic peptide segments with the sequences of Gly-Ser-Hyp-Gly-Gly-Hyp-Gly-Ala-Ala-Gly-Phe-Hyp-Gly-Arg, gly-Leu-Ala-Gly-Pro-Hyp-Gly-Met-Hyp-Gly-Ala-Arg and Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Asp-Hyl through a large number of experimental screens, and the 3 elk characteristic peptide segments have high specificity and can be used for distinguishing elk, hippomk and sika deer horns. The elk horn characteristic peptide fragment and the detection method thereof provided by the invention screen out the optimal chromatographic condition and mass spectrum condition through a large number of experiments, the whole method is simple to operate and accurate to judge, and horn medicinal materials from closely related species, namely elk horn, red deer horn and plum blossom deer horn can be accurately distinguished. The elk horn characteristic peptide segment and the detection method thereof provided by the invention are beneficial to the quality control of elk horns and have important significance for ensuring the quality of elk horn-containing products.
Description
Technical Field
The invention relates to a method for detecting and identifying animal drugs, in particular to elk horn characteristic peptide and a detection method thereof.
Background
Elk population is growing, the picking-up amount of elk horn is increasing year by year, and the problem of medicinal status recovery is receiving more and more attention. In recent years, elk horn has been studied for its effects of nourishing yin, resisting aging and depression, but there has been no research on its quality standard.
The method provides reference and basis for elk antler quality research, is favorable for further perfecting the quality standard of elk antler and products thereof, provides theoretical reference for the formulation of the quality standard of elk antler medicinal materials, lays a foundation for the industrial development of elk antler medicinal resources, and provides scientific basis for clinical medication thereof.
Disclosure of Invention
The main purpose of the invention is to provide a characteristic peptide segment of elaeagnus davidianus and a method for detecting the characteristic peptide segment of elaeagnus davidianus in horn animal drugs, aiming at the defects. The elk horn characteristic peptide provided by the invention has strong specificity. The provided method is simple to operate and high in sensitivity, can accurately identify whether the horn animal medicine contains the elk horn component, and provides a scientific method for ensuring the quality of the elk horn and products thereof.
The technical scheme is as follows: in order to realize the purpose, the invention adopts the technical scheme that:
the elk horn characteristic peptide segment has the sequence shown in the sequence table 1:
characteristic peptide fragment 1: gly-Ser-Hyp-Gly-Gly-Hyp-Gly-Ala-Ala-Gly-Phe-Hyp-Gly-Gly-Arg;
characteristic peptide fragment 2: gly-Leu-Ala-Gly-Pro-Hyp-Gly-Met-Hyp-Gly-Ala-Arg;
characteristic peptide fragment 3: gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Asp-Hyl;
internal reference peptide segment: gly-Glu-Thr-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val-Gly-Ala-Arg.
A method for detecting elk horn characteristic peptide fragments comprises the following steps:
(1) Preparing a reference substance solution from a characteristic peptide segment 1, a characteristic peptide segment 2 and an internal reference peptide segment of elk horn;
(2) After extracting protein from a horn animal drug sample to be detected, performing enzyme digestion by using protease, injecting an enzymolysis solution and the reference substance solution obtained in the step (1) into a liquid chromatograph-mass spectrometer, taking the elk horn characteristic peptide segment as a reference, adopting a multi-reaction monitoring mode, and selecting the conditions as follows: characteristic peptide fragment 1: m/z 645.3 double charge → 573.3, dp =80.00, ce =35.00, propeptide stretch 2: m/z 556.8, double charge → 407.8, dp =78.51, ce =29.36, propeptide stretch 3: m/z 804.40 double charge → 448.2, dp =144.01, CE =37.80, reference peptide stretch: m/z 781.4 double charge → 556.3, DP =91.00, CE =41.40, and the elk horn, the hippophae horn and the sika horn are distinguished according to the ion detection.
If the ion pair of the peptide segment 1 is detected, the retention time of the ion is consistent with the elk horn characteristic peptide segment 1 reference substance, and the daughter ion of the ion is consistent with the daughter ion of the reference substance, the elk horn component is contained in the sample to be detected; all the elk antler, the antler of horsetail and the antler of sika deer can detect the characteristic peptide segment 2, wherein, the elk antler A Characteristic peptide-2 /A Internal reference peptide The value of is not less than 0.182, cornu Cervi is Characteristic peptide-2 /A Internal reference peptide Has a value of not higher than 0.044, and Cervus Nippon Temminck et Schlegel A Characteristic peptide-2 /A Internal reference peptide A value of not higher than 0.047;
all-grass of elk horn A Characteristic peptide-3 /A Internal reference peptide The value of is not less than 2.038, and cornu Cervi is Characteristic peptide-3 /A Internal reference peptide The value of (A) is not higher than 0.497, and cornu Cervi is Characteristic peptide-3 /A Internal reference peptide Is not higher than 0.555.
Preferably, the elk horn characteristic peptide fragment detection method is carried out as follows: taking 20mg of horn animal medicine sample to be detected, adding 1ml of protein lysate, homogenizing for 3 times, centrifuging, taking supernatant, adding cold acetone solution, fully and uniformly mixing, refrigerating, and precipitating overnight;
centrifuging an acetone precipitate sample, removing a supernatant, washing the precipitate with cold acetone once, volatilizing the precipitate, adding a urea solution for redissolving, fully shaking, adding a Tris-HCl buffer solution after the precipitate is completely dissolved, adding trypsin into each horn sample respectively, and incubating in a constant-temperature water bath to obtain the compound.
Preferably, the elk horn characteristic peptide fragment detection method is carried out as follows: taking 20mg of the horn-like animal drug sample to be tested, adding 1ml of a protein lysis solution comprising 4% SDS,20mMDTT,50mM Tris-HCl, pH =8.8; adding grinding beads, homogenizing for 3 times, centrifuging at 60s,12000rpm for 20min each time, measuring protein content by BCA, taking 1000 μ g protein, adding 80% cold acetone solution with 5.5 times of sample solution volume, mixing well, and freezing and precipitating at-20 deg.C overnight;
taking an acetone sediment sample, centrifuging at 12000rpm for 20min, discarding the supernatant, washing the sediment twice by using 80% cold acetone, volatilizing the sediment, adding 200 mu l of 8M urea solution for redissolving, fully shaking, adding 1400 mu l of Tris-HCl buffer solution after the sediment is completely dissolved, reducing the urea concentration to below 1M, respectively adding 40 mu l of trypsin of 0.1 mu g/mu l into each angle sample, and incubating in a constant-temperature water bath at 37 ℃ for 12h to obtain the composition.
Preferably, in the method for detecting elaeagnus davurica characteristic peptide fragment, 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 having a size of 2.1 μm × 50mm, a loading amount of 5 μ l, a flow rate of 0.3ml/min, a mobile phase A of 0.1% formic acid, a mobile phase B of acetonitrile, 0 to 3.5min,5 to 20% B linear gradient elution, 3.5 to 4min,20 to 5% B linear gradient elution, 4 to 6min,5% B isocratic elution; triple quadrupole mass spectrometry, electrospray positive ion mode ESI +, mass spectrometry parameters are as follows: the ion source temperature is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60psi; the ion source gas 2 is supplied to the ion source,60psi。
the ion pair in MRM mode is: characteristic peptide fragment 1: m/z 645.3 double charge → 573.3, dp =80.00, ce =35.00, propeptide stretch 2: m/z 556.8 double charge → 407.8, dp =78.51, ce =29.36, signature peptide stretch 3: m/z 804.40 double charge → 448.2, dp =144.01, ce =37.80, internal reference peptide fragment: m/z 781.4 double charge → 556.3, dp =91.00, CE =41.40.
Preferably, the horn animal includes elk horn, horsehorn and sika horn.
Has the advantages that: the invention screens out 2 elk horn characteristic peptide segments and internal reference peptide segments through a large number of experimental screens, and the 2 elk horn characteristic peptide segments have high specificity and can be used for identifying medicines containing elk horn medicinal materials.
The elk horn characteristic peptide segment and the detection method thereof provided by the invention screen out the optimal chromatographic condition and mass spectrum condition through a large number of experiments. The whole method is simple to operate and accurate in judgment, and the elk horn characteristic peptide segments can be accurately distinguished. The elk horn characteristic peptide segment and the detection method thereof provided by the invention are beneficial to the quality control of elk horn medicines and have important significance for ensuring the quality of elk horn-containing medicines.
Drawings
FIG. 1 is XIC diagram of elk horn characteristic peptide segments 1-3;
FIG. 2 is XIC diagram of elk horn 3 characteristic peptides and internal reference peptide segment;
FIG. 3 is XIC diagram of 3 characteristic peptides and internal reference peptide segments of Cervus elaphus Linnaeus;
FIG. 4 is XIC diagram of 3 characteristic peptides and internal reference peptide segments of Cervus nippon Temminck.
Detailed Description
The invention is further described in the following examples in connection with specific embodiments thereof, it is to be understood that these examples are included solely for the purpose of illustration and are not intended as limitations on the scope of the invention, as modifications of various equivalent forms of the invention which would occur to those skilled in the art upon reading the present disclosure are intended to be within the scope of the invention as defined in the appended claims.
Example 1
The elk horn characteristic peptide segment has the sequence shown in the sequence table 1:
characteristic peptide fragment 1: gly-Ser-Hyp-Gly-Gly-Gly-Hyp-Gly-Ala-Ala-Gly-Phe-Hyp-Gly-Gly-Arg;
characteristic peptide fragment 2: gly-Leu-Ala-Gly-Pro-Hyp-Gly-Met-Hyp-Gly-Ala-Arg;
characteristic peptide fragment 3: gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Asp-Hyl;
reference peptide fragment: gly-Glu-Thr-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val-Gly-Ala-Arg.
EXAMPLE 2 detection of characteristic peptide fragment 1 in various keratinaceous animal drugs
(1) 20mg of each of elk antler, horsehorn antler and sika antler was added to 1ml of protein lysate [4% SDS,20mM DTT,50mM Tris-HCl (pH = 8.8) ]]Homogenizing for 3 times, centrifuging at 60s,12000rpm for 20min, measuring protein content by BCA, respectively collecting 1000 μ g protein from cornu Cervi Elaphus davidianus, cornu Cervi Pantotrichum, and cornu Cervi Degelatinatum, respectively adding 25 μ l 0.2M IAA solution, standing in dark for 30min, adding 5.5 times of V Sample(s) The solution is mixed well and frozen at-20 ℃ for precipitation overnight.
(2) And (2) respectively taking the acetone precipitation sample in the step (1), centrifuging at 12000rpm for 20min, discarding supernatant, washing the precipitate once with 80% cold acetone, volatilizing the precipitate, adding 200 mu l of 8M urea solution for redissolving, fully shaking, adding 1400 mu l of Tris-HCl buffer solution after the precipitate is completely dissolved to reduce the urea concentration to be less than 1M, respectively adding 40 mu l of trypsin with the concentration of 0.1 mu g/mu l into the elk antler, the red antler and the sika antler samples, and incubating in constant-temperature water bath at 37 ℃ for 12h to obtain elk antler, red antler and sika antler enzymolysis liquid.
(3) The pure product of the characteristic peptide fragment 1 described in example 1 is prepared into 1ml of control solution with the concentration of 200 ng/ml.
(4) Detecting each horn enzymolysis liquid in the step (2) and the characteristic peptide fragment 1 reference substance solution in the step (3) by adopting a liquid chromatograph-mass spectrometer, wherein the liquid phase conditions are as follows: the chromatographic column is 1.7 mu m Waters C 18 Column (2.1 μm. Times.50 mm), loading 5 μ l, flow rate 0.3ml/min, mobile phase A0.1% formic acid, mobile phase B acetonitrile, 0-3.5min, 5-20% B-linearGradient elution, 3.5-4 min, 20-5% B Linear gradient elution, 4-6 min,5% B isocratic elution. Triple quadrupole mass spectrometry is adopted, and the mass spectrometry parameters are as follows: the ion source temperature is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60psi; ion source gas 2, 60psi. The ion pairs for the MRM mode are: m/z 645.3 (double charge) → 573.3 (DP =80.00, CE = 35.00).
The results are shown in FIG. 1, in which (A) is a control of characteristic peptide fragment 1; (B) is a characteristic peptide fragment 2 reference substance; (C) The elk horn sample can detect an ion peak consistent with the characteristic peptide fragment 1 control solution as a characteristic peptide fragment 3 control, and no ion peak is detected in other samples, so that the elk horn sample can specifically detect the characteristic peptide fragment 1 component in the elk horn, thereby being distinguished from the hippophae horn and the sika horn.
EXAMPLE 3 determination of the relative content of characteristic Polypeptides in various Keratin-based drugs
(1) The elk antler, the deer horn and the sika antler protein extract are prepared by respectively taking a proper amount of coarse powder of the elk antler, the deer horn or the sika antler, adding 10 times of water for decocting for 6 hours, filtering, continuously adding water into medicine residues, repeatedly decocting for 2 times, combining the three extraction filtrates, concentrating, and freeze-drying to respectively obtain the lyophilized powder of the elk antler, the deer horn and the sika antler protein extract.
(2) 20mg of each of the 10 batches of elk antler, 10 batches of red antler and 10 batches of sika antler protein extracts prepared according to the method of step (1) were added to 1ml of each protein lysis solution [4% SDS,2 mM DTT,50mM Tris-HCl (pH = 8.8) ]]Homogenizing for 3 times, centrifuging at 60s,12000rpm for 20min each time, measuring protein content by BCA, collecting 1000 μ g protein, adding 25 μ l 0.2M IAA solution, standing in dark for 30min, adding 5.5 times of V Sample (I) The solution is mixed well and frozen at-20 ℃ for precipitation overnight.
(3) And (3) respectively taking the acetone precipitation samples in the step (2), centrifuging at 12000rpm for 20min, discarding the supernatant, washing the precipitate once with 80% cold acetone, volatilizing the precipitate, adding 200 mu l of 8M urea solution for redissolving, fully shaking, adding 1400 mu l of Tris-HCl buffer solution after the precipitate is completely dissolved to reduce the urea concentration to below 1M, respectively adding 40 mu l of trypsin of 0.1 mu g/mu l into each angle sample, and incubating in a constant-temperature water bath at 37 ℃ for 12h to obtain the enzyme-hydrolyzed solution of each angle.
(4) The pure products of the characteristic peptide fragment 1, the characteristic peptide fragment 2, the characteristic peptide fragment 3 and the internal reference peptide fragment described in the example 1 are prepared into 1ml of mixed reference solution with the concentration of 200 ng/ml.
(5) Detecting each angle enzymolysis liquid in the step (3) and the characteristic peptide section 1, the characteristic peptide section 2, the characteristic peptide section 3 and the reference peptide section reference substance solution in the step (4) by adopting a liquid chromatography-mass spectrometer, wherein the liquid phase conditions are as follows: the chromatographic column is 1.7 mu m Waters C 18 Column (2.1 μm. Times.50 mm), loading 10 μ l, flow rate 0.3ml/min, mobile phase A0.1% formic acid, mobile phase B acetonitrile, 0-3.5min, 5-20% B linear gradient elution, 3.5-4 min, 20-5% B linear gradient elution, 4-6 min,5% B isocratic elution. Triple quadrupole mass spectrometry is adopted, and the mass spectrometry parameters are as follows: the ion source temperature is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60psi; ion source gas 2, 60psi. The ion pairs for the MRM mode are: characteristic peptide fragment 1: m/z 645.3 (double charge) → 573.3 (DP =80.00, ce = 35.00), signature peptide stretch 2: m/z 556.8 (double charge) → 407.8 (DP =78.51, ce = 29.56), propeptide stretch 3: m/z 804.40 double charge → 448.2, DP =144.01, ce =37.80, reference peptide fragment: m/z 781.4 (double charge) → 556.3 (DP =91.00, ce = 41.40).
The results are shown in Table 1, and no characteristic peptide 1 was detected in 10 batches of antler from horse and 10 batches of antler from sika. Peak area ratio of characteristic peptide 2 of elk antler to internal reference peptide (A) of 10 batches Characteristic peptide-2 /A Internal reference peptide ) Average value of 0.1822; peak area ratio of 10 batches of antler-like characteristic peptide 2 to internal reference peptide (A) Characteristic peptide-2 /A Internal reference peptide ) The average value is 0.0445, and the peak area ratio of the characteristic peptide 2 of 10 batches of sika deer antler to the internal reference peptide (A) Characteristic peptide-2 /A Internal reference peptide ) The average value was 0.0479.
Peak area ratio of characteristic peptide 3 of elk horn to internal reference peptide (A) of 10 batches Characteristic peptide-3 /A Internal reference peptide ) The average value is 2.0387; 10. peak area ratio of characteristic peptide 3 of batch of cornu cervi to internal reference peptide (A) Characteristic peptide-3 /A Internal reference peptide ) Average value of 0.4973Peak area ratio of 10 batches of spotted deer horn characteristic peptide 3 to internal reference peptide (A) Characteristic peptide-3 /A Internal reference peptide ) The average value was 0.5556.
From the characteristic peptide ratio, elk deer horn, horsedeer horn and sika deer horn can be distinguished.
TABLE 1 Peak areas and ratios of characteristic peptide fragments of elk antler, deer antler and plum blossom antler
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Nanjing university of traditional Chinese medicine
<120> elk horn characteristic peptide segment and detection method thereof
<141> 2022-05-06
<160> 4
<170> SIPOSequenceListing 1.0
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<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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Gly Ser Gly Gly Gly Ala Ala Gly Phe Gly Gly Arg
1 5 10
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<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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Gly Leu Ala Gly Pro Gly Met Gly Ala Arg
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<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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Gly Leu Thr Gly Pro Ile Gly Pro Gly Pro Ala Gly Ala Gly Asp
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<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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Gly Glu Thr Gly Pro Ala Gly Pro Ile Gly Pro Val Gly Ala Arg
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Claims (10)
1. An elk horn characteristic peptide segment, which is characterized in that:
characteristic peptide fragment 1: gly-Ser-Hyp-Gly-Gly-Gly-Hyp-Gly-Ala-Ala-Gly-Phe-Hyp-Gly-Gly-Arg;
characteristic peptide fragment 2: gly-Leu-Ala-Gly-Pro-Hyp-Gly-Met-Hyp-Gly-Ala-Arg;
characteristic peptide fragment 3: gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Asp-Hyl;
internal reference peptide segment: gly-Glu-Thr-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val-Gly-Ala-Arg.
2. The detection method of the elk horn characteristic peptide segment is characterized by comprising the following steps:
(1) Preparing a control solution from the 3 elk antler characteristic peptide fragments and the internal reference peptide fragments of claim 1;
(2) Extracting protein from a horn animal drug sample to be detected, then carrying out enzyme digestion by using protease, respectively injecting an enzymolysis solution and 3 elk horn characteristic peptide section and internal reference peptide section reference substance solutions in the step (1) into a liquid mass spectrometer, taking the elk horn characteristic peptide section as a reference, and adopting a multi-reaction monitoring mode, wherein the selected MRM conditions are as follows: characteristic peptide fragment 1: m/z 645.3 double charge → 573.3, dp =80.00, ce =35.00, propeptide stretch 2: m/z 556.8 double charge → 407.8, dp =78.51, ce =29.36, propeptide stretch 3: m/z 804.40 double charge → 448.2, dp =144.01, ce =37.80, internal reference peptide fragment: m/z 781.4 double charge → 556.3, DP =91.00, CE =41.40, and the different horn animal drug samples are distinguished according to the ion detection.
3. The method for detecting elaeagnus davidianus characteristics peptide fragment according to claim 2, characterized in that if peptide fragment 1 ion pair is detected, the retention time of the ion is consistent with that of elaeagnus davidianus characteristics peptide fragment 1 reference substance, and the daughter ion is consistent with that of the reference substance, elaeagnus davidianus component is contained in the sample to be detected; the cornu Elaphani davidianus, cornu Cervi Pantotrichum and cornu Cervi may be used for detecting characteristic peptide segment 2 and characteristic peptide segment 3, wherein cornu Elaphani davidianus A Characteristic peptide-2 /A Internal reference peptide The value of is not less than 0.182, cornu Cervi is Characteristic peptide-2 /A Internal reference peptide Has a value of not higher than 0.044, and Cervus Nippon Temminck et Schlegel A Characteristic peptide-2 /A Internal reference peptide A value of not higher than 0.047;
all-grass of elk horn A Characteristic peptide-3 /A Internal reference peptide The value of (A) is not less than 2.038, and cornu Cervi is Characteristic peptide-3 /A Internal reference peptide The value of (A) is not higher than 0.497, and cornu Cervi is Characteristic peptide-3 /A Internal reference peptide Is not higher than 0.555.
4. The method for detecting elk horn characteristic peptide fragment according to claim 2, wherein the horn animal drug sample protein extraction method is: taking a proper amount of horn animal medicine samples to be detected, adding 10 times of water, extracting under reflux for 6 hours, repeating for 3 times, combining filtrates, concentrating, and freeze-drying to obtain elk antler extract.
5. The method for detecting elk horn-like characteristic peptide fragments according to claim 2, wherein the enzyme digestion method comprises water bath digestion or ultrasonic digestion by using trypsin, pepsin, elastase, acid protease, alkaline protease or complex enzyme.
6. The method for detecting elk horn characteristic peptide segment according to claim 2, characterized in that the extraction method of horn animal drug sample protein is: taking 20mg of elk antler sample to be detected, adding 1ml of protein lysate, homogenizing for 3 times, centrifuging, taking supernatant, determining protein content, taking 1000 mu g of protein, adding cold acetone solution, fully mixing, refrigerating, and precipitating overnight; centrifuging an acetone precipitate sample, removing a supernatant, washing the precipitate with cold acetone once, volatilizing the precipitate, adding a urea solution for redissolving, fully shaking, adding a Tris-HCl buffer solution after the precipitate is completely dissolved, adding trypsin into each horn sample respectively, and incubating in a constant-temperature water bath to obtain the compound.
7. The method for detecting elk horn characteristic peptide segment according to claim 6, characterized in that the enzyme digestion method is carried out as follows: taking 20mg of the horn drug sample to be detected, and adding 1ml of a protein lysate containing 4% SDS,2 mM DTT,50mM Tris-HCl, pH =8.8; adding grinding beads, homogenizing for 3 times, centrifuging at 60s,12000rpm for 2 min each time, measuring protein content by BCA, taking 1000 μ g of protein, adding 80% cold acetone solution with 5.5 times of sample volume, mixing completely, and freezing and precipitating at-20 deg.C overnight;
taking an acetone sediment sample, centrifuging at 12000rpm for 20min, discarding the supernatant, washing the sediment once with 80% cold acetone, volatilizing the sediment, adding 200 mu l of 8M urea solution for redissolving, fully shaking, adding 1400 mu l of Tris-HCl buffer solution after the sediment is completely dissolved, reducing the urea concentration to below 1M, respectively adding 40 mu l of trypsin of 0.1 mu g/mu l into each angle sample, and incubating in a constant-temperature water bath at 37 ℃ for 12h to obtain the composition.
8. The method for detecting elk horn characteristic peptide segment according to claim 2, characterized in that the enzyme digestion method is carried out as follows: collecting 20mg of cornu Cervi Pantotrichum extract, adding 50ml of 1% ammonium bicarbonate solution, performing ultrasonic treatment for 30min to fully dissolve the cornu Cervi extract, centrifuging at 10000rpm, collecting 100 μ l of cornu Cervi extraction solution, placing in 2ml centrifuge tube, adding 20 μ l of 0.1 μ g/μ l trypsin solution, shaking, and performing enzymolysis at 37 deg.C for 12 hr.
9. The method for detecting elk horn characteristic peptide fragment according to claim 2, characterized in that 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, having a size of 2.1 μm × 50mm, a loading amount of 5 μ l, a flow rate of 0.3ml/min, a mobile phase A of 0.1% formic acid, a mobile phase B of acetonitrile, 0 to 3.5min,5 to 20% B linear gradient elution, 3.5 to 4min,20 to 5% B linear gradient elution, 4 to 6min,5% B isocratic elution; triple quadrupole mass spectrometry is adopted, and the mass spectrometry parameters are as follows: the ion source temperature is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60psi; ion source gas 2, 60psi; the ion pair in MRM mode is: characteristic peptide fragment 1: m/z 645.3, double charge → 573.3, dp =80.00, ce =35.00, signature peptide stretch 2: m/z 556.8, double charge → 407.8, dp =78.51, ce =29.36, propeptide stretch 3: m/z 804.40 double charge → 448.2, dp =144.01, ce =37.80, internal reference peptide fragment: m/z 781.4 double charge → 556.3, dp =91.00, ce =41.40.
10. The method for detecting the elk horn characteristic peptide fragment according to claim 2, wherein the horn-type animal includes elk horn, red horn, sika horn, caribou horn, moose horn or water deer horn.
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| CN117147881B (en) * | 2023-10-30 | 2024-01-02 | 山东省食品药品检验研究院 | Deer antler characteristic peptide fragment, method for detecting deer antler and application |
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