A peptide segment characteristic of eld's deer horn and a detection method thereof

Abstract

The application discloses a characteristic peptide segment of eld's deer horn and a detection method thereof. The application screens out three characteristic peptide segments of eld's deer horn, namely, Gly-Ser-Hyp-Gly-Gly-Hyp-Gly-Ala-Ala-Gly-Phe-Hyp-Gly-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 experiments. The three characteristic peptide segments of eld's deer horn have high specificity and can be used for distinguishing eld's deer horn, red deer horn and sika deer horn. The characteristic peptide segment of eld's deer horn and the detection method thereof provided by the application screen out optimal chromatographic conditions and mass spectrometry conditions through a large number of experiments. The whole method is simple in operation, accurate in judgment and capable of accurately distinguishing horn medicines from close species, namely, eld's deer horn, red deer horn and sika deer horn. The characteristic peptide segment of eld's deer horn and the detection method thereof provided by the application are favorable for quality control of eld's deer horn and have important significance for guaranteeing the quality of products containing eld's deer horn.

Description

Technical Field

[0001] This invention relates to a method for detecting and identifying animal drugs, specifically to characteristic peptides of elk antlers and their detection methods. Background Technology

[0002] As the population of Père David's deer continues to grow, the amount of antlers collected is increasing year by year, and the restoration of its medicinal value is receiving increasing attention. In recent years, there have been many studies on the effects of Père David's deer antlers in nourishing yin, anti-aging, and anti-depression, but there is still no research on its quality standards.

[0003] This invention detects the components of Père David's antler by detecting two characteristic peptide segments. This method provides a reference and basis for the quality research of Père David's antler, which is conducive to the further improvement of the quality standards of Père David's antler and its products, provides a theoretical reference for the formulation of quality standards for Père David's antler medicinal materials, lays the foundation for the industrialization of medicinal resources of Père David's antler, and provides a scientific basis for its clinical use. Summary of the Invention

[0004] The main objective of this invention is to address the aforementioned shortcomings by providing a method for detecting characteristic peptides of elk antlers in antler-based animal medicines, specifically elk antler peptides. The elk antler peptides provided by this invention exhibit high specificity. The provided method is simple to operate, highly sensitive, and can accurately identify whether elk antler components are present in antler-based animal medicines, thus providing a scientific method for ensuring the quality of elk antlers and their products.

[0005] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0006] A characteristic peptide segment of elk antler, the sequence of which is shown in Sequence Listing 1:

[0007] Characteristic peptide 1:

[0008] Gly-Ser-Hyp-Gly-Gly-Hyp-Gly-Ala-Ala-Gly-Phe-Hyp-Gly-Gly-Arg;

[0009] Characteristic peptide 2: Gly-Leu-Ala-Gly-Pro-Hyp-Gly-Met-Hyp-Gly-Ala-Arg;

[0010] Characteristic peptide 3:

[0011] Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Asp-Hyl;

[0012] Internal reference peptide: Gly-Glu-Thr-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val-Gly-Ala-Arg.

[0013] A method for detecting characteristic peptides in elk antlers, comprising the following steps:

[0014] (1) Prepare a reference solution by mixing characteristic peptide 1, characteristic peptide 2 and internal reference peptide from elk antler;

[0015] (2) After extracting proteins from the antler-derived drug samples to be tested, the samples were digested with protease. The digest and the control solution from step (1) were then injected into a liquid chromatography-mass spectrometry (LC-MS) instrument. Using the characteristic peptides of elk antlers as controls, multiple reaction monitoring (MRM) was employed with the following conditions: Characteristic peptide 1: m / z 645.3 double charge → 573.3, DP = 80.00, CE = 35.00; Characteristic peptide 2: m / z 556.8, double charge → 407.8, DP = 78.51, CE = 29.36; Characteristic peptide 3: m / z 804.40 double charge → 448.2, DP = 144.01, CE = 37.80; Internal control peptide: m / z 781.4 double charge → 556.3, DP=91.00, CE=41.40, detection was performed, and the antlers of elk, red deer, and sika deer were distinguished based on the detected ion conditions.

[0016] If peptide 1 ion pair is detected, and the retention time of the ion is consistent with that of the elk antler characteristic peptide 1 reference standard, and its daughter ion is consistent with that of the reference standard, then the sample to be tested contains elk antler component; characteristic peptide 2 can be detected in elk antler, red deer antler, and sika deer antler, wherein, elk antler A 特征肽-2 / A 内参肽 The value is not less than 0.182, sika deer antler A 特征肽-2 / A 内参肽 The value is not higher than 0.044, sika deer antler A 特征肽-2 / A 内参肽 The value is no higher than 0.047;

[0017] Elk Antlers A 特征肽-3 / A 内参肽 The value is not less than 2.038, sika deer antler A 特征肽-3 / A 内参肽 The value is not higher than 0.497, sika deer antler A 特征肽-3 / A 内参肽 The value is no higher than 0.555.

[0018] As a preferred embodiment, the enzyme digestion method for detecting the characteristic peptides of elk antlers described above is as follows: Take 20 mg of the antler animal drug sample to be tested, add 1 ml of protein lysis buffer, homogenize 3 times, centrifuge, take the supernatant, add cold acetone solution, mix thoroughly, refrigerate, and let it precipitate overnight.

[0019] After centrifuging the acetone precipitate sample, discard the supernatant, wash the precipitate once with cold acetone, evaporate the precipitate, add urea solution to reconstitute, shake thoroughly, and after the precipitate is completely dissolved, add Tris-HCl buffer. Add trypsin to each horn sample and incubate in a constant temperature water bath to obtain the final product.

[0020] As a preferred embodiment, the enzyme digestion method for detecting the characteristic peptides of elk antlers described above is as follows: Take 20 mg of the antler-derived drug sample to be tested, add 1 ml of protein lysis buffer, which includes 4% SDS, 20 mM DTT, 50 mM Tris-HCl, pH = 8.8; add grinding beads, homogenize 3 times, 60 s each time, centrifuge at 12000 rpm for 20 min, determine the protein content by BCA, take 1000 μg of protein, add 5.5 times the sample solution volume of 80% cold acetone solution, mix thoroughly, and freeze at -20℃ overnight for precipitation;

[0021] Take the acetone precipitate sample, centrifuge at 12000 rpm for 20 min, discard the supernatant, wash the precipitate twice with 80% cold acetone, evaporate the precipitate, add 200 μl of 8M urea solution to reconstitute, shake thoroughly, and after the precipitate is completely dissolved, add 1400 μl of Tris-HCl buffer to reduce the urea concentration to below 1M. Add 40 μl of 0.1 μg / μl trypsin to each horn sample, and incubate in a constant temperature water bath at 37℃ for 12 h to obtain the final product.

[0022] As a preferred embodiment, the liquid chromatography conditions for detecting the characteristic peptides of elk antlers described above using liquid chromatography-mass spectrometry (LC-MS) are as follows: 1.7 μm Waters C10 column. 18 The column was 2.1 μm × 50 mm, the sample loading volume was 5 μl, the flow rate was 0.3 mL / min, the mobile phase A was 0.1% formic acid, and the mobile phase B was acetonitrile. Elution was performed with a linear gradient of 5% B from 0 to 3.5 min, linear gradient of 20% B from 3.5 to 4 min, and isocratic elution of 5% B from 4 to 6 min. Triple quadrupole mass spectrometry was used in positive ion electrospray ionization mode (ESI+). The mass spectrometry parameters were: ion source temperature 500 °C; ionization voltage 5500 V; desolventizing temperature 500 °C; ion source gas 1, 60 psi; ion source gas 2, 60 psi.

[0023] The ion pairs in MRM mode are: Characteristic peptide 1: m / z 645.3 double charge → 573.3, DP = 80.00, CE = 35.00; Characteristic peptide 2: m / z 556.8 double charge → 407.8, DP = 78.51, CE = 29.36; Characteristic peptide 3: m / z 804.40 double charge → 448.2, DP = 144.01, CE = 37.80; Internal reference peptide: m / z 781.4 double charge → 556.3, DP = 91.00, CE = 41.40.

[0024] As a preferred option, the detection method for characteristic peptides of elk antlers described above applies to antler-based animal drugs, including elk antlers, red deer antlers, and sika deer antlers.

[0025] Beneficial effects: Through extensive experimental screening, this invention has identified two characteristic peptide segments and an internal reference peptide segment of elk antler. These two characteristic peptide segments of elk antler have high specificity and can be used to identify medicines containing elk antler medicinal materials.

[0026] The elk antler characteristic peptides and their detection method provided by this invention utilizes optimal chromatographic and mass spectrometric conditions determined through extensive experimental screening. The entire method is simple to operate, accurate in judgment, and can precisely distinguish elk antler characteristic peptides. The elk antler characteristic peptides and their detection method provided by this invention are beneficial for the quality control of elk antler pharmaceutical products and are of great significance in ensuring the quality of pharmaceutical products containing elk antler. Attached Figure Description

[0027] Figure 1 XIC diagram of characteristic peptide segments 1-3 of elk antler;

[0028] Figure 2 XIC plots of three characteristic peptides and internal reference peptides from elk antler;

[0029] Figure 3 XIC plots of three characteristic peptides and internal reference peptides from red deer antler;

[0030] Figure 4 XIC plot of three characteristic peptides and internal reference peptides from sika deer antler. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0032] Example 1

[0033] A characteristic peptide segment of elk antler, the sequence of which is shown in Sequence Listing 1:

[0034] Characteristic peptide 1:

[0035] Gly-Ser-Hyp-Gly-Gly-Hyp-Gly-Ala-Ala-Gly-Phe-Hyp-Gly-Gly-Arg;

[0036] Characteristic peptide 2: Gly-Leu-Ala-Gly-Pro-Hyp-Gly-Met-Hyp-Gly-Ala-Arg;

[0037] Characteristic peptide 3:

[0038] Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Asp-Hyl;

[0039] Internal reference peptide: Gly-Glu-Thr-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val-Gly-Ala-Arg.

[0040] Example 2: Detection of Characteristic Peptide 1 in Various Keratin-Based Animal Drugs

[0041] (1) Take 20 mg of each of the elk antler, red deer antler, and sika deer antler samples, add 1 ml of protein lysis buffer [4% SDS, 20 mM DTT, 50 mM Tris-HCl (pH = 8.8)], homogenize 3 times, 60 s each time, centrifuge at 12000 rpm for 20 min, and determine the protein content by BCA. Take 1000 μg of protein from each of the elk antler, red deer antler, and sika deer antler samples, add 25 μl of 0.2 M IAA solution, incubate in the dark for 30 min, and add 5.5 times the amount of vitamin C. 样品 Mix thoroughly with an 80% cold acetone solution and freeze at -20°C overnight to allow precipitation.

[0042] (2) Take the acetone precipitate samples from step (1), centrifuge at 12000 rpm for 20 min, discard the supernatant, wash the precipitate once with 80% cold acetone, evaporate the precipitate, add 200 μl of 8M urea solution to reconstitute, shake thoroughly, and add 1400 μl of Tris-HCl buffer to reduce the urea concentration to below 1M after the precipitate is completely dissolved. Add 40 μl of 0.1 μg / μl trypsin to the elk antler, red deer antler and sika deer antler samples respectively, and incubate in a constant temperature water bath at 37℃ for 12 h to obtain elk antler, red deer antler and sika deer antler enzymatic hydrolysate.

[0043] (3) Prepare 1 ml of a reference solution with a concentration of 200 ng / ml by dissolving the pure product of characteristic peptide 1 described in Example 1.

[0044] (4) The enzymatic hydrolysates from step (2) and the characteristic peptide 1 reference solution from step (3) were analyzed using liquid chromatography-mass spectrometry (LC-MS). The LC conditions were: 1.7 μm Waters C10 column. 18 The column (2.1 μm × 50 mm) was used, with a sample loading volume of 5 μl and a flow rate of 0.3 mL / min. Mobile phase A was 0.1% formic acid, and mobile phase B was acetonitrile. Elution was performed using a linear gradient of 5% to 20% B from 0 to 3.5 min, a linear gradient of 20% to 5% B from 3.5 to 4 min, and isocratic elution of 5% B from 4 to 6 min. Triple quadrupole mass spectrometry was used with the following parameters: ion source temperature 500 °C; ionization voltage 5500 V; desolventizing temperature 500 °C; ion source gas 1, 60 psi; ion source gas 2, 60 psi. The ion pair in MRM mode was: m / z 645.3 (double charge) → 573.3 (DP = 80.00, CE = 35.00).

[0045] See results Figure 1 In the figure, (A) is the reference standard for characteristic peptide 1; (B) is the reference standard for characteristic peptide 2; and (C) is the reference standard for characteristic peptide 3. The ion peaks consistent with the reference standard solution for characteristic peptide 1 can be detected in the elk antler sample, while they are not detected in other samples. This indicates that the present invention can specifically detect the characteristic peptide 1 component in elk antler, thereby distinguishing it from red deer antler and sika deer antler.

[0046] Example 3: Determination of the relative content of characteristic polypeptides in various keratin-based animal drugs

[0047] (1) Preparation of protein extracts from elk antlers, red deer antlers and sika deer antlers: Take appropriate amounts of coarse powder from elk antlers, red deer antlers or sika deer antlers respectively, add 10 times the amount of water and decoct for 6 hours, filter, add water to the residue and repeat the decoction twice, combine the three extracts, concentrate, freeze dry, and obtain freeze-dried powders of protein extracts from elk antlers, red deer antlers and sika deer antlers respectively.

[0048] (2) Take 20 mg of protein extract samples from 10 batches of elk antlers, 10 batches of red deer antlers, and 10 batches of sika deer antlers prepared according to step (1), add 1 ml of protein lysis buffer [4% SDS, 20 mM DTT, 50 mM Tris-HCl (pH = 8.8)], homogenize 3 times, 60 s each time, centrifuge at 12000 rpm for 20 min, determine the protein content by BCA, take 1000 μg of protein, add 25 μl of 0.2 M IAA solution, place in the dark for 30 min, and add 5.5 times the amount of vitamin C. 样品 Mix thoroughly with an 80% cold acetone solution and freeze at -20°C overnight to allow precipitation.

[0049] (3) Take the acetone precipitate samples from step (2), centrifuge at 12000 rpm for 20 min, discard the supernatant, wash the precipitate once with 80% cold acetone, evaporate the precipitate, add 200 μl of 8M urea solution to reconstitute, shake thoroughly, and after the precipitate is completely dissolved, add 1400 μl of Tris-HCl buffer to reduce the urea concentration to below 1M. Add 40 μl of 0.1 μg / μl trypsin to each horn sample, and incubate in a constant temperature water bath at 37℃ for 12 h to obtain the horn enzyme hydrolysate.

[0050] (4) Prepare a mixed reference solution of 1 ml with a concentration of 200 ng / ml by dissolving the pure characteristic peptides 1, 2, 3 and internal reference peptides described in Example 1.

[0051] (5) The enzymatic hydrolysates from step (3) and the characteristic peptide 1, characteristic peptide 2, characteristic peptide 3, and internal reference peptide standard solutions from step (4) were analyzed using liquid chromatography-mass spectrometry (LC-MS). The LC conditions were: 1.7 μm Waters C10 column. 18 The column (2.1 μm × 50 mm) was used, with a sample loading volume of 10 μl and a flow rate of 0.3 mL / min. Mobile phase A was 0.1% formic acid, and mobile phase B was acetonitrile. Elution was performed using a linear gradient of 5% to 20% B from 0 to 3.5 min, a linear gradient of 20% to 5% B from 3.5 to 4 min, and isocratic elution of 5% B from 4 to 6 min. Triple quadrupole mass spectrometry was used with the following parameters: ion source temperature 500 °C; ionization voltage 5500 V; desolventizing temperature 500 °C; ion source gas 1, 60 psi; ion source gas 2, 60 psi. The ion pairs in MRM mode are: Characteristic peptide 1: m / z 645.3 (double charge) → 573.3 (DP = 80.00, CE = 35.00), Characteristic peptide 2: m / z 556.8 (double charge) → 407.8 (DP = 78.51, CE = 29.56), Characteristic peptide 3: m / z 804.40 (double charge) → 448.2, DP = 144.01, CE = 37.80, and Internal reference peptide: m / z 781.4 (double charge) → 556.3 (DP = 91.00, CE = 41.40).

[0052] The results are shown in Table 1. Characteristic peptide 1 was not detected in any of the 10 batches of red deer antler and 10 batches of sika deer antler. The peak area ratio (A) of characteristic peptide 2 to the internal reference peptide in the 10 batches of elk antler is shown in Table 1. 特征肽-2 / A 内参肽 The average value was 0.1822; the peak area ratio of characteristic peptide 2 to internal reference peptide in 10 batches of sika deer antler (A) 特征肽-2 / A 内参肽 The average value was 0.0445. The peak area ratio of characteristic peptide 2 to internal reference peptide in 10 batches of sika deer antler (A) 特征肽-2 / A 内参肽The average value is 0.0479.

[0053] The peak area ratio of elk antler characteristic peptide 3 to internal reference peptide in 10 batches (A 特征肽-3 / A 内参肽 The average value was 2.0387; the peak area ratio of characteristic peptide 3 to internal reference peptide in 10 batches of sika deer antler (A) 特征肽-3 / A 内参肽 The average value was 0.4973. The peak area ratio of characteristic peptide 3 to internal reference peptide in 10 batches of sika deer antler (A) 特征肽-3 / A 内参肽 The average value is 0.5556.

[0054] The antlers of elk, red deer, and sika deer can be distinguished by their characteristic peptide ratios.

[0055] Table 1. Peak areas and ratios of characteristic peptides from elk antlers, red deer antlers, and sika deer antlers.

[0056]

[0057]

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A group of moose horn characteristic peptide segments, characterized in that, The characteristic peptide segment group is: Characteristic peptide segment 1: Gly-Ser-Hyp-Gly-Gly-Hyp-Gly-Ala-Ala-Gly-Phe-Hyp-Gly-Gly-Arg; Characteristic peptide segment 2: Gly-Leu-Ala-Gly-Pro-Hyp-Gly-Met-Hyp-Gly-Ala-Arg; Characteristic peptide segment 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. A method for detecting a characteristic peptide segment of a moose horn, characterized by, The method comprises the following steps: (1) preparing a control solution of the three characteristic peptide segments and the internal reference peptide segment of the elk horn according to claim 1; (2) extracting the protein from the sample of the horn animal medicine to be detected, and then performing enzyme cutting with a protease, and then injecting the enzyme solution and the control solution of the three characteristic peptide segments and the internal reference peptide segment of the elk horn in step (1) into a liquid chromatograph-mass spectrometer, taking the characteristic peptide segments of the elk horn as a control, adopting a multiple reaction monitoring mode, and selecting MRM conditions as follows: characteristic peptide segment 1: m / z 645.3 double charge→573.3, DP=80.00, CE=35.00, characteristic peptide segment 2: m / z 556.8 double charge→407.8, DP=78.51, CE=29.36, characteristic peptide segment 3: m / z 804.40 double charge→448.2, DP=144.01, CE=37.80, internal reference peptide segment: m / z 781.4 double charge→556.3, DP=91.00, CE=41.40, and performing detection, and different horn animal medicine samples are distinguished according to the detection of the above ions; If the peptide segment 1 ion pair is detected, and the retention time of the ion is consistent with that of the characteristic peptide segment 1 control product, and the daughter ion is consistent with that of the control product, then the sample to be detected contains the elk horn component; the elk horn, the red deer horn and the sika deer horn can all detect the characteristic peptide segment 2 and the characteristic peptide segment 3, wherein the value of the elk horn A characteristic peptide-2 / A internal reference peptide is not less than 0.182, the value of the red deer horn A characteristic peptide-2 / A internal reference peptide is not higher than 0.044, and the value of the sika deer horn A characteristic peptide-2 / A internal reference peptide is not higher than 0.047; The value of the elk horn A characteristic peptide-3 / A internal reference peptide is not less than 2.038, the value of the red deer horn A characteristic peptide-3 / A internal reference peptide is not higher than 0.497, and the value of the sika deer horn A characteristic peptide-3 / A internal reference peptide is not higher than 0.555; The enzyme cutting method is water bath enzyme cutting or ultrasonic enzyme cutting using trypsin; The horn animal medicine is the elk horn, the red deer horn or the sika deer horn.

3. The method for detecting characteristic peptides of elk antlers according to claim 2, characterized in that, The protein extraction method of the horn animal medicine sample is as follows: an appropriate amount of the horn animal medicine sample to be detected is taken, 10 times water is added, and backflow extraction is performed for 6 hours, and the operation is repeated for 3 times, the filtrates are combined, concentrated, and freeze-dried to obtain the elk horn extract.

4. The method of detecting moose antler characteristic peptide segments according to claim 2, characterized in that, The protein extraction method of the sample of the antlered animal medicine is as follows: 20 mg of the sample of the antlered animal medicine to be detected is added with 1 ml of protein lysate, and homogenized for 3 times, centrifuged, and the supernatant is taken to determine the protein content, 1000 μg of protein is taken, and added with cold acetone solution, mixed thoroughly, and then refrigerated and precipitated overnight; the acetone precipitated sample is centrifuged, and the supernatant is discarded, the precipitate is washed with cold acetone once, the precipitate is dried, added with urea solution to dissolve, shaken thoroughly, and then added with Tris-HCl buffer after the precipitate is completely dissolved, and 0.1 μg / μl of trypsin is added in each sample of the antlered animal, and then incubated in a constant temperature water bath to obtain the sample.

5. The method of detecting moose antler characteristic peptide segments according to claim 4, characterized in that, The enzyme digestion method is as follows: 20 mg of the sample of the antlered animal medicine to be detected is added with 1 ml of protein lysate, and the protein lysate includes 4% SDS, 20 mM DTT, 50 mM Tris-HCl, and pH=8.8; grinding beads are added, and homogenized for 3 times, 60 s each time, and centrifuged at 12000 rpm for 20 min, and the protein content is determined by BCA, 1000 μg of protein is taken, and added with 5.5 times the sample volume of 80% cold acetone solution, mixed thoroughly, and then refrigerated and precipitated overnight at-20℃; The acetone precipitated sample is centrifuged at 12000 rpm for 20 min, and the supernatant is discarded, the precipitate is washed with 80% cold acetone once, the precipitate is dried, added with 200 μl of 8M urea solution to dissolve, shaken thoroughly, and then added with 1400 μl of Tris-HCl buffer after the precipitate is completely dissolved, so that the urea concentration is reduced to below 1M, 0.1 μg / μl of trypsin 40 μl is added in each sample of the antlered animal, and then incubated in a constant temperature water bath at 37℃ for 12 hours to obtain the sample.

6. The method of detecting moose antler characteristic peptide segments according to claim 2, characterized in that, The enzyme digestion method is as follows: 20 mg of the sample of the antlered animal medicine to be detected is added with 1% ammonium bicarbonate solution 50 ml, and ultrasonically treated for 30 minutes to dissolve the sample of the antlered animal, and then centrifuged at 10000 rpm, 100 μl of the sample of the antlered animal is taken and placed in a 2 ml centrifuge tube, 0.1 μg / μl of trypsin solution 20 μl is added, shaken, and then incubated in a constant temperature water bath at 37℃ for 12 hours to obtain the sample.

7. The method for detecting characteristic peptides of elk antlers according to claim 2, characterized in that, The liquid chromatography-mass spectrometry instrument detection liquid phase condition is: the chromatographic column is 1.7 μm Waters C 18 column, the size is 2.1 μm×50 mm, the sample loading amount is 5 μl, the flow rate is 0.3 ml / min, the mobile phase A is 0.1% formic acid, the mobile phase B is acetonitrile, 0-3.5 min, 5-20% B linear gradient elution, 3.5-4 min, 20-5% B linear gradient elution, 4-6 min, 5% B isocratic elution; three quadrupole mass spectrometer is used, and the mass spectrometry parameter is: ion source temperature 500 DEG C; ionization voltage 5500 V; desolvation temperature 500 DEG C; ion source gas 1, 60 psi; ion source gas 2, 60 psi; the ion pair of MRM mode is: characteristic peptide segment 1: m / z 645.3, double charge→573.3, DP=80.00, CE=35.00, characteristic peptide segment 2: m / z 556.8, double charge→407.8, DP=78.51, CE=29.36, characteristic peptide segment 3: m / z 804.40 double charge→448.2, DP=144.01, CE=37.80, internal reference peptide segment: m / z 781.4 double charge→556.3, DP=91.00, CE=41.40.

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