WO2022048049A1 - Characteristic peptide segment capable of distinguishing deer horn gelatin from deer skin gelatin, and test method therefor - Google Patents

Abstract

A characteristic peptide segment capable of distinguishing a deer horn gelatin from a deer skin gelatin, and a test method therefor. By screening through a large number of experiments, a graph is drawn by using the proportion of a deer horn gelatin in a mixed gelatin as an abscissa and using values of A_{peptide 1}/A_{peptide 3}, and A_{peptide 2}/A_{peptide 3} as ordinates, the proportion of the deer horn gelatin has a linear relationship to A_{peptide 1}/A_{peptide 3}, and a standard curve equation is established as: y=1.034x+0.0157, R²=0.9908; the proportion of the deer horn gelatin also has a linear relationship to A_{peptide 2}/A_{peptide 3}, and a standard curve equation is established as: y=0.7883x+0.0949, R ²=0.9905; the proportion of the deer horn gelatin doped is related to the values of A_{peptide 1}/A_{peptide 3}, and A_{peptide 2}/A_{peptide 3}, and a mixing proportion of the deer horn gelatin and a deer skin gelatin can be determined according to the ratios of A_{peptide 1}/A_{peptide 3}, and A_{peptide 2}/A_{peptide 3}. The method is strong in specificity, high in sensitivity and simple in operation, and is capable of distinguishing the deer horn gelatin from the deer skin gelatin, and controlling the quality.

Description

A characteristic peptide segment capable of distinguishing staghorn glue and deerskin glue and its detection method technical field

The invention relates to a deer-derived characteristic peptide segment and a detection method thereof, in particular to a characteristic peptide segment and detection method for distinguishing deerskin glue from deerskin glue, as well as a sample mixed with deerskin glue and the proportion of deerskin glue mixed in the deer skin glue method.

technical background

Glue-like medicinal materials include donkey-hide gelatin, deerskin glue, antler glue, yellow gelatin, etc. More than 80% of the ingredients are different types of collagen, including type I collagen α1 chain (COL1A1), type I collagen α2 chain (COL1A2), type II collagen Collagen α1 chain (COL2A1), type III collagen α1 chain (COL3A1), etc., among which peptides derived from type I collagen (COL1) are the main ones. As a highly conserved protein, COL1 widely exists in different animal species, and is one of the important protein components that constitute glue-like medicinal materials.

Both deer antler glue and deerskin glue are derived from sika deer or red deer, both of which are precious Chinese medicinal materials. Deer antler glue is a solid glue made of sika deer or red deer antlers by boiling and concentrating, and deerskin glue is sika deer or red deer. The dried or fresh skins are boiled and concentrated into solid glue. Deer antlers are ossified horns of red deer or sika deer, and the price is much higher than that of deer skin. There is a phenomenon that deerskin glue is mixed with deer antler glue to make shoddy products. How to distinguish deerskin glue from deerskin glue is really a difficult problem in the identification and research of glue-like medicinal materials, which brings challenges to the identification of deerskin glue and deerskin glue, as well as the identification of counterfeit products mixed with deerskin glue and deerskin glue.

Deer antler glue and deerskin glue are indistinguishable in appearance, and both are derived from sika deer or red deer, and their protein composition is the same. Therefore, it is basically impossible to distinguish between the two by looking for species-specific peptides.

SUMMARY OF THE INVENTION

Purpose of the invention: The present invention studies and determines the ratio of the relative contents of three deer-derived peptide segments through a large number of experimental screenings, so as to be used to distinguish deerskin glue and deer antler glue. The method has strong specificity, high sensitivity and simple operation, and can be used for the differentiation and quality control of deerskin glue and antler glue.

To achieve the above object, the present invention adopts the following technical solutions:

A characteristic peptide segment capable of distinguishing antler gum and deerskin gum, wherein the characteristic peptide is:

Peptide 1:

Gly-Ser-Asp-Gly-Ser-Val-Gly-Pro-Val-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Ser-Ala-Gly-Pro-Hyp-Gly-Phe-Hyp-Gly- Ala-Hyp-Gly-Pro-Lys

Peptide 2:

Gly-Ser-Asp-Gly-Ser-Val-Gly-Pro-Val-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Ser-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly- Ala-Hyp-Gly-Pro-Lys

Peptide 3:

Gly-Asp-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly- Ala-Val-Gly-Ala-Lys.

A method for detecting a characteristic peptide segment capable of distinguishing antler glue and deerskin glue, is characterized in that, comprises the following steps:

(1) 3 deer-derived characteristic peptides described in claim 1 are made into mixed reference solution;

(2) After the deerskin glue and deer antler glue samples to be detected are digested with trypsin, the enzymolyzed solution and the mixed reference solution of the deer source characteristic peptide in step (1) are injected into the liquid mass spectrometer. Peptide control, using ESI positive ion mode, multiple reaction monitoring mode, the selected transitions include: peptide 1: m/z 864.0 (triple charge) → 535.4, peptide 2: m/z 858.7 (triple charge) → 527.4 for detection, Peptide 3: m/z 845.4 (three charges) → 507.4 for detection; the ratio of peptide ₁ peak area A to peptide 3 peak area A _{peptide 3} or the ratio of peptide ₂ peak area A to peptide 3 peak area A to _{peptide 3} The ratio of , determines whether the sample is buckskin glue or staghorn glue.

As a preferred solution, the above-mentioned detection method for distinguishing characteristic peptide segments of staghorn glue and deerskin glue, the enzymatic cleavage method is as follows: take 10 mg of the glue-like medicinal material sample to be detected, add 5 ml of phosphate buffer (pH= 6.0～8.5), ultrasonically dissolve the sample completely, centrifuge at 12000rpm for 20min, take 150μl of the supernatant, put it in a 2ml centrifuge tube, dilute it with 1ml 50mM PBS, add an appropriate amount of trypsin, shake well, fully enzymolysis, add 10% v 60 μl of aqueous solution of /v trifluoroacetic acid to terminate the reaction, centrifuge at 12,000 rpm for 20 min to obtain the enzymatic hydrolysate of glue-like medicinal materials, and store at -20°C for later use.

As a preferred solution, in the above-mentioned method for detecting characteristic peptides that can distinguish between staghorn glue and deerskin glue, the mass concentration of trypsin added is 0.1% to 10%.

As a preferred solution, the above-mentioned detection method for distinguishing characteristic peptides of staghorn glue and deerskin glue, the enzymatic hydrolysis methods include: 37 ℃ constant temperature enzymatic hydrolysis, 500W-1000W microwave-assisted enzymatic hydrolysis, 20kHz-100kHz ultrasonic-assisted enzymatic hydrolysis , any one or a combination of enzyme immobilization and enzymolysis.

As a preferred solution, the above-mentioned detection method for distinguishing characteristic peptide segments of staghorn gum and buckskin gum, the liquid phase conditions detected by LC/MS are as follows: the chromatographic column is a 1.7 μm Waters C ₁₈ column, and the specification is 2.1 μm ×100mm, sample volume is 2μl, flow rate is 0.3ml/min, 0～3.5min, 10～30%A linear gradient elution, 3.5～4min, 30～10%A linear gradient elution, 4～6min, 10%A A elution; using triple quadrupole mass spectrometry, the mass spectrometry conditions are: m/z 864.0 (triple charge)→535.4, m/z 858.7 (triple charge)→527.4, m/z 845.4 (triple charge)→507.4.

The A _{-peptide 1} /A _{-peptide 3} of pure antler glue shall not be lower than 1.0, the A _{-peptide 2} /A _{-peptide 3} shall not be lower than 0.5, the A _{-peptide 1} /A _{-peptide 3} of pure buckskin glue shall not be higher than 0.2, and the A _{-peptide 2} /A _{peptide 3} must not be higher than 0.5. More preferably, the A _{-peptide 1} /A _{-peptide 3} of pure antler glue shall not be lower than 1.3, the A _{-peptide 2} /A _{-peptide 3} shall not be lower than 0.7, and the A _{-peptide 1} /A _{-peptide 3} of pure deerskin glue shall not be higher than 0.08 , A _{peptide 2} /A _{peptide 3} should not be higher than 0.19.

As a preferred version, the above-described detection method of the characteristic peptide segment of staghorn glue and staghorn glue can be distinguished, and the establishment of the standard curve equation for the detection of the ratio contained in staghorn glue and staghorn glue:

The antler glue and deerskin glue were mixed in the proportions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively. 5ml of phosphate buffered saline (pH=6.0～8.5), the sample was completely dissolved by ultrasound, centrifuged at 12000rpm for 20min, 150μl of supernatant was taken, placed in a 2ml centrifuge tube, diluted with 1ml of 50mM PBS, and added with a mass concentration of 1% trypsin , shake well, microwave enzymolysis for 30min, after enzymolysis, add 60 μl of 10% v/v trifluoroacetic acid aqueous solution to terminate the reaction, centrifuge at 12000rpm for 20min to obtain the enzymolysis solution of mixed gel samples in different proportions, store at -20°C for later use ;

The enzymatic hydrolyzate solution of the mixed gel samples in different proportions was injected into the LC/MS, and the injection volume was 1 μg. The liquid phase conditions detected by the LC/MS were as follows: the chromatographic column was a 1.7 μm C ₁₈ reversed-phase chromatographic column, and the size was 2.1 μm× 100mm, flow rate 0.3ml/min, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid, 0~3.5min, 10~30%A linear gradient elution, 3.5~4min, 30~10%A linear gradient elution , 4～6min, 10%A elution;

The mass spectrometry conditions detected by LC-MS were: electrospray positive ion mode ESI+, mass spectrometry parameters were: ion source temperature 500°C; ionization voltage 5500V; desolvation temperature 500°C; ion source gas 1, 60 psi; ion source gas 2, 60psi;

The transition conditions corresponding to the characteristic peptides are set as follows:

Peptide 1: m/z 864.0 (triple charge)→535.4, DP=166.57, CE=33.95;

Peptide 2: m/z 858.7 (triple charge)→527.4, DP=160.35, CE=35.65;

Peptide 3: m/z 845.4 (triple charge)→507.4, DP=143.27, CE=31.50.

Taking the proportion of antler gum in the mixed glue as the abscissa and the values of A _{peptide 1} /A _{peptide 3} and A _{peptide 2} /A _{peptide 3} as the ordinate, the proportion of antler gum is linear with A _{peptide 1} /A _{peptide 3} , the established standard curve equation is y=1.034x+0.0157, R ² =0.9908; the proportion of staghorn gum and A _{peptide 2} /A _{peptide 3} are also linear, the established standard curve equation is y=0.7883x+0.0949, R ² =0.9905 The proportion of antler gum is related to the values of A _{peptide 1 /} A _{peptide 3 ,} A _{peptide 2 /} A _{peptide 3} , and the ratio of A peptide 1/A peptide 3 and A peptide 2/A peptide 3 can be used to determine the ratio of antler gum and deerskin. glue mixing ratio.

A detection kit for identifying antler glue and deerskin glue, the kit comprises the three characteristic peptide segments of claim 1. Of course, other reagents necessary for liquid and mass detection can also be included as needed.

In the present invention, whether the sample to be tested is pure staghorn glue or pure staghorn glue can be determined by the above method and the above-mentioned kit, and the ratio of stagskin glue and staghorn glue in the mixed glue can also be determined.

Beneficial effect: the present invention has the following advantages compared with the prior art:

The invention researches and determines the ratio of the relative contents of three deer-derived peptide segments through a large number of experimental screenings, and establishes a linear equation for distinguishing deerskin glue and antler glue. The method has strong specificity, high sensitivity and simple operation, and can be used for the differentiation and quality control of deerskin glue and antler glue. The invention can overcome the deficiencies in the prior art that the antler glue and the deerskin glue are difficult to distinguish in appearance, and the exclusive peptide segment is also difficult to distinguish, and very good technical progress is achieved.

Description of drawings

Figure 1 shows the relationship between the proportion of antler gum in the mixed glue and the values of A _{peptide 1} /A _{peptide 3} and A _{peptide 2} /A _{peptide 3} .

FIG. 2 is a mass spectrum of peptide 1. FIG.

FIG. 3 is a mass spectrum of peptide 2. FIG.

FIG. 4 is a mass spectrum of peptide 3. FIG.

specific embodiment

The present invention will be further described below in conjunction with specific embodiments, but these embodiments should not be construed as limiting the present invention.

Trypsin used in the following examples was purchased from Promega.

Example 1

A deer-derived characteristic peptide, the characteristic peptide sequence has 3, such as sequence table 1:

Peptide 1:

Gly-Ser-Asp-Gly-Ser-Val-Gly-Pro-Val-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Ser-Ala-Gly-Pro-Hyp-Gly-Phe-Hyp-Gly- Ala-Hyp-Gly-Pro-Lys;

Peptide 2:

Gly-Ser-Asp-Gly-Ser-Val-Gly-Pro-Val-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Ser-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly- Ala-Hyp-Gly-Pro-Lys;

Peptide 3:

Gly-Asp-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly- Ala-Val-Gly-Ala-Lys.

The above-mentioned polypeptides were prepared by Nanjing GenScript Biotechnology Co., Ltd. by solid-phase synthesis.

Example 2 Determination of deerskin glue A _{-peptide 1} /A _{-peptide 3} and A _{-peptide 2} /A _{-peptide 3}

Take 10 batches of commercially available deerskin glue samples, 10 mg of each batch, add 5 ml of phosphate buffer (pH=7.8), ultrasonically dissolve the samples, centrifuge at 12,000 rpm for 20 min, take 150 μl of the supernatant, and put it in 2 ml In a centrifuge tube, dilute with 1ml 50mM PBS, add 1wt% trypsin, shake well, enzymolysis at a constant temperature of 37°C for 12h, add 60μl of 10%v/v TFA aqueous solution after enzymolysis to terminate the reaction, and centrifuge at 12000rpm for 20min to obtain deerskin glue. The enzymatic hydrolysate was stored at -20°C for later use.

The above 10 batches of deerskin glue enzymatic hydrolyzed solution were injected into the LC/MS, and the injection volume was 1 μg. The liquid phase conditions detected by the LC/MS were as follows: the chromatographic column was a 1.7 μm C18 reversed-phase chromatographic column (2.1 μm ×100mm), flow rate 0.3ml/min, mobile phase A (acetonitrile), mobile phase B (0.1% formic acid), 0～3.5min, 10～30%A linear gradient elution, 3.5～4min, 30～10%A Linear gradient elution, 4 ~ 6min, 10% A elution. The mass spectrometry conditions detected by LC-MS were: electrospray positive ion mode (ESI+), and the mass spectrometry parameters were: ion source temperature 500°C; ionization voltage 5500V; desolvation temperature 500°C; ion source gas 1, 60 psi; ion source gas 2, 60psi. The transition conditions corresponding to the characteristic peptides are set as follows:

Peptide 1: m/z 864.0 (triple charge)→535.4, DP=166.57, CE=33.95;

Peptide 2: m/z 858.7 (triple charge)→527.4, DP=160.35, CE=35.65;

Peptide 3: m/z 845.4 (triple charge)→507.4, DP=143.27, CE=31.50.

The values of 10 batches of deerskin glue A _{peptide 1} /A _{peptide 3} and A _{peptide 2} /A _{peptide 3} are shown in Table 1. The average value of A _{peptide 1} /A _{peptide 3} is 0.085±0.039, and the average value of A _{peptide 2} /A _{peptide 3} is 0.085±0.039. is 0.186±0.099.

Table 1 Deerskin glue A _{-peptide 1} /A _{-peptide 3} , A _{-peptide 2} /A _{-peptide 3} results

Example 3 Determination of antler gum A _{peptide 1} /A _{peptide 3} and A _{peptide 2} /A _{peptide 3}

Take 10 batches of antler samples, prepare antler gum according to the 2020 edition of "Chinese Pharmacopoeia" to obtain antler gum samples, take about 10 mg of each batch, add 5 ml of phosphate buffer (pH=7.8), and ultrasonically dissolve the samples completely , centrifuge at 12000rpm for 20min, take 150μl of supernatant, put it in a 2ml centrifuge tube, dilute with 1ml 50mM PBS, add 1wt% trypsin, shake well, ultrasonicate for 10min, add 60μl of 10% v/v TFA aqueous solution after enzymolysis The reaction was terminated and centrifuged at 12,000 rpm for 20 min to obtain the deer carrageenan enzymatic hydrolysate, which was stored at -20°C for later use.

10 batches of deer caratum enzymatic hydrolysate were injected into the LC/MS, and the injection volume was 1 μg. The liquid phase conditions detected by the LC/MS were as follows: the chromatographic column was a 1.7 μm C18 reversed-phase chromatographic column (2.1 μm × 100mm), flow rate 0.3ml/min, mobile phase A (acetonitrile), mobile phase B (0.1% formic acid), 0～3.5min, 10～30%A linear gradient elution, 3.5～4min, 30～10%A linear Gradient elution, 4-6 min, 10% A elution. The mass spectrometry conditions detected by LC-MS were: electrospray positive ion mode (ESI+), and the mass spectrometry parameters were: ion source temperature 500°C; ionization voltage 5500V; desolvation temperature 500°C; ion source gas 1, 60 psi; ion source gas 2, 60psi. The transition conditions corresponding to the characteristic peptides are set as follows:

Peptide 1: m/z 864.0 (triple charge)→535.4, DP=166.57, CE=33.95;

Peptide 2: m/z 858.7 (triple charge)→527.4, DP=160.35, CE=35.65;

Peptide 3: m/z 845.4 (triple charge)→507.4, DP=143.27, CE=31.50.

The values of A _{peptide 1} /A _{peptide 3} and A _{peptide 2} /A _{peptide 3} of 10 batches of antler gum are shown in Table 2. The average value of A _{peptide 1} /A _{peptide 3} is 1.659±0.453, and the average value of A _{peptide 2} /A _{peptide 3} is 0.898±0.167.

Table 2 Deer carat A _{peptide 1} /A _{peptide 3} , A _{peptide 2} /A _{peptide 3} results

Example 4 Determination of A _{-peptide 1} /A _{-peptide 3} and A _{-peptide 2} /A _{-peptide 3} in mixed samples of deerskin glue and antler glue in different proportions

Mix the antler glue and deerskin glue according to the ratio of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and mix each batch of glue Take 10 mg of each sample, add 5 ml of phosphate buffered saline (pH=7.8), sonicate to completely dissolve the sample, centrifuge at 12,000 rpm for 20 min, take 150 μl of the supernatant, put it in a 2 ml centrifuge tube, dilute it with 1 ml of 50 mM PBS, add 1 wt% pancreatic Protease, shake well, microwave enzymolysis for 30min, add 60 μl of 10% v/v TFA aqueous solution after enzymolysis to terminate the reaction, centrifuge at 12,000rpm for 20min to obtain enzymolysis solutions of mixed gel samples in different proportions, and store at -20°C for later use.

The enzymatic hydrolyzed solution of the mixed gel samples in different proportions was injected into the LC-MS in turn, and the sample injection amount was 1 μg. ), flow rate 0.3ml/min, mobile phase A (acetonitrile), mobile phase B (0.1% formic acid), 0～3.5min, 10～30%A linear gradient elution, 3.5～4min, 30～10%A linear gradient Elution, 4 ~ 6min, 10% A elution.

The mass spectrometry conditions detected by LC-MS were: electrospray positive ion mode (ESI+), and the mass spectrometry parameters were: ion source temperature 500°C; ionization voltage 5500V; desolvation temperature 500°C; ion source gas 1, 60 psi; ion source gas 2, 60psi. The transition conditions corresponding to the characteristic peptides are set as follows:

Peptide 1: m/z 864.0 (triple charge)→535.4, DP=166.57, CE=33.95;

Peptide 2: m/z 858.7 (triple charge)→527.4, DP=160.35, CE=35.65;

Peptide 3: m/z 845.4 (triple charge)→507.4, DP=143.27, CE=31.50.

The values of A _{-peptide 1} /A _{-peptide 3} and A _{-peptide 2} /A _{-peptide 3} of the mixed gel samples with different ratios are shown in Table 3. The proportion of antler gum in the mixed gel is the same as that of A _{-peptide 1} /A _{-peptide 3} , A _{-peptide 2} /A _{-peptide 3} The numerical relationship is shown in Figure 1. Taking the proportion of antler gum in the mixed glue as the abscissa and the values of A _{peptide 1} /A _{peptide 3} and A _{peptide 2} /A _{peptide 3} as the ordinate, the proportion of antler gum is linear with A _{peptide 1} /A _{peptide 3} , y=1.034x+0.0157, R ² =0.9908; the proportion of staghorn gum is also linear with A _{peptide 2} /A _{peptide 3} , y=0.7883x+0.0949, R ² =0.9905. It shows that the proportion of antler gum is related to the values of A peptide ₁ /A peptide ₃ and A peptide ₂ /A _peptide 3, so the ratio of A _{peptide 1} /A _{peptide 3} and A _{peptide 2} /A _{peptide 3} can be used to determine the ratio of antler gum and deer Leather glue mixing ratio.

Table 3 The relationship between the blending ratio and the peak area of the antler gum/deerskin gum mixed sample

Example 5:

Take a certain concentration of peptide 1, peptide 2 and peptide 3 mixed reference substance, inject 6 times continuously under the aforementioned chromatographic-mass spectrometry conditions, measure the peak area of peptide 1, peptide 2 and peptide 3 reference substance, and calculate the peak area of the reference substance. RSD, the results are shown in Table 4, peptide 1, peptide 2 and peptide 3 were 2.00%, 0.83% and 2.70%, respectively, indicating that the precision of the method was good.

Taking the corresponding concentration when the signal-to-noise ratio (S/N) of peptide 1, peptide 2 and peptide 3 is about 3 as the limit of detection (LOD), the signal-to-noise ratio (S/N) of peptide 1, peptide 2 and peptide 3 is about 10 The corresponding concentration is the limit of detection (LOQ). The results are shown in Table 4. The LOQ and LOD of peptide 1 are 0.62 and 0.21 ng/ml, respectively; the LOQ and LOD of peptide 2 are 0.67 and 0.22 ng/ml, respectively; the LOQ of peptide 3 and LOD of 0.58 and 0.19 ng/ml, respectively.

Table 4 Precision, detection limit and quantification limit of peptide 1, peptide 2 and peptide 3

	Precision (RSD,%)	LOQ(ng/ml)	LOD(ng/ml)
Peptide 1	2.00	0.62	0.21
Peptide 2	0.83	0.67	0.22
Peptide 3	2.70	0.58	0.19

Claims (9)

1. A characteristic peptide segment capable of distinguishing antler gum and deerskin gum, wherein the characteristic peptide is:

   Peptide 1:

   Gly-Ser-Asp-Gly-Ser-Val-Gly-Pro-Val-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Ser-Ala-Gly-Pro-Hyp-Gly-Phe-Hyp-Gly- Ala-Hyp-Gly-Pro-Lys

   Peptide 2:

   Gly-Ser-Asp-Gly-Ser-Val-Gly-Pro-Val-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Ser-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly- Ala-Hyp-Gly-Pro-Lys

   Peptide 3:

   Gly-Asp-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly- Ala-Val-Gly-Ala-Lys.
2. A method for detecting a characteristic peptide segment capable of distinguishing antler glue and deerskin glue, is characterized in that, comprises the following steps:

   (1) 3 deer-derived characteristic peptides described in claim 1 are made into mixed reference solution;

   (2) After the deerskin glue and deer antler glue samples to be detected are digested with trypsin, the enzymolyzed solution and the mixed reference solution of the deer source characteristic peptide in step (1) are injected into the liquid mass spectrometer. Peptide control, using ESI positive ion mode, multiple reaction monitoring mode, the selected transitions include: peptide 1: m/z 864.0 (triple charge) → 535.4, peptide 2: m/z 858.7 (triple charge) → 527.4 for detection, Peptide 3: m/z 845.4 (three charges) → 507.4 for detection; the ratio of peptide ₁ peak area A to peptide 3 peak area A _{peptide 3} or the ratio of peptide ₂ peak area A to peptide 3 peak area A to _{peptide 3} The ratio of , determines whether the sample is buckskin glue or staghorn glue.
3. The detection method for distinguishing characteristic peptide segments of staghorn glue and deerskin glue according to claim 2, characterized in that, the enzymatic cleavage method is: taking 10 mg of a sample of glue-like medicinal materials to be detected, adding 5 ml of phosphate buffer solution (PBS), ultrasonicated to dissolve the sample completely, centrifuged at 12,000 rpm for 20 min, took 150 μl of the supernatant, placed it in a 2 ml centrifuge tube, diluted with 1 ml of 50 mM PBS, added an appropriate amount of trypsin, shaken well, fully enzymatically hydrolyzed, and added 10% trifluoride The reaction was terminated with 60 μl of acetic acid (TFA) solution, and centrifuged at 12,000 rpm for 20 min to obtain an enzymatic hydrolysate of glue-like medicinal materials, which was stored at -20° C. for later use.
4. The method for detecting characteristic peptide segments that can distinguish staghorn glue and deerskin glue according to claim 3, wherein the mass concentration of the trypsin added is 0.1% to 10%.
5. The detection method for distinguishing characteristic peptide segments of staghorn glue and deerskin glue according to claim 3, wherein the enzymatic hydrolysis method comprises: 37°C constant temperature enzymatic hydrolysis, microwave-assisted enzymatic hydrolysis, ultrasonic-assisted enzymatic hydrolysis, and enzyme immobilization enzymatic hydrolysis.
6. The detection method for distinguishing characteristic peptide segments of staghorn glue and deerskin glue according to claim 3, is characterized in that, the liquid phase condition detected by liquid mass spectrometer is: chromatographic column is 1.7 μm Waters C ₁₈ column, specification is 2.1μm×100mm, the loading volume is 2μl, the flow rate is 0.3ml/min, 0～3.5min, 10～30%A linear gradient elution, 3.5～4min, 30～10%A linear gradient elution, 4～6min , 10% A elution; using triple quadrupole mass spectrometry, mass spectrometry conditions are: m/z 864.0 (triple charge)→535.4, m/z 858.7 (triple charge)→527.4, m/z 845.4 (triple charge)→507.4 .
7. The detection method for distinguishing characteristic peptide segments of staghorn glue and deerskin glue according to claim 2, wherein the A _{peptide 1} /A _{peptide 3} of the staghorn glue must not be lower than 1.0, and the A _{peptide 2} /A _{peptide 3} must not be lower than 1.0. Below 0.5, the A _{-peptide 1} /A _{-peptide 3} of deerskin glue shall not be higher than 0.2, and the A _{-peptide 2} /A _{-peptide 3} shall not be higher than 0.5.
8. The detection method of the characteristic peptide segment that can distinguish staghorn glue and staghorn glue according to claim 2, is characterized in that, the establishment of the standard curve equation of the ratio detection of staghorn glue and staghorn glue:

   Mix the antler glue and deerskin glue according to the ratio of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and mix each batch of glue Take 10 mg of each sample, add 5 ml of phosphate buffered saline, sonicate to completely dissolve the sample, centrifuge at 12,000 rpm for 20 min, take 150 μl of the supernatant, put it in a 2 ml centrifuge tube, dilute it with 1 ml of 50 mM PBS, and add 1% trypsin. Shake well, microwave enzymolysis for 30min, add 60 μl of 10% trifluoroacetic acid aqueous solution after enzymolysis to terminate the reaction, and centrifuge at 12000rpm for 20min to obtain enzymolyzed solutions of mixed gel samples in different proportions, which are stored at -20°C for later use;

   The enzymatic hydrolyzate solution of the mixed gel samples in different proportions was injected into the LC/MS, and the injection volume was 1 μg. The liquid phase conditions detected by the LC/MS were as follows: the chromatographic column was a 1.7 μm C ₁₈ reversed-phase chromatographic column, and the size was 2.1 μm× 100mm, flow rate 0.3ml/min, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid, 0~3.5min, 10~30%A linear gradient elution, 3.5~4min, 30~10%A linear gradient elution , 4～6min, 10%A elution;

   The mass spectrometry conditions detected by LC-MS were: electrospray positive ion mode ESI+, and the mass spectrometry parameters were: ion source temperature 500 °C; ionization voltage 5500 V; desolvation temperature 500 °C; ion source gas 1, 60 psi; ion source gas 2, 60psi;

   The transition conditions corresponding to the characteristic peptides are set as follows:

   Peptide 1: m/z 864.0 (triple charge)→535.4, DP=166.57, CE=33.95;

   Peptide 2: m/z 858.7 (triple charge)→527.4, DP=160.35, CE=35.65;

   Peptide 3: m/z 845.4 (triple charge)→507.4, DP=143.27, CE=31.50.

   Taking the proportion of antler gum in the mixed glue as the abscissa and the values of A _{peptide 1} /A _{peptide 3} and A _{peptide 2} /A _{peptide 3} as the ordinate, the proportion of antler gum is linear with A _{peptide 1} /A _{peptide 3} , the established standard curve equation is y=1.034x+0.0157, R2=0.9908; the proportion of antler gum and A _{peptide 2} /A _{peptide 3} are also linear, the established standard curve equation is y=0.7883x+0.0949, R2=0.9905; The ratio of glue incorporation is related to the values of A _{peptide 1 /} A _{peptide 3 and} A _{peptide 2 /} A _{peptide 3.} The ratio of A peptide 1 /A peptide 3 and A peptide 2 /A peptide 3 can be used to determine the mixture of staghorn glue and deerskin glue. Proportion.
9. A detection kit for identifying antler glue and deerskin glue, characterized in that the kit comprises the three characteristic peptide segments of claim 1 .

Images