Character polypeptide for distinguishing and identifying antler medicinal material based on chemical marker, kit and identification method thereof

Abstract

The invention belongs to the field of biological detection, and particularly relates to a marker polypeptide for distinguishing and identifying antler medicinal materials and preparations thereof based on chemical markers, a kit and an identification method of the marker polypeptide. According to the chemical marker adopted by the invention, chemical substances which are specific from a certain medicinal material (decoction piece) but not from other medicinal materials are searched as identification indexes and are used for true and false identification and quality control of the traditional Chinese medicine. The marker polypeptide is used as a chemical marker of the antler medicinal material, so that a plurality of species can be distinguished and identified. The used chemical marker-marker polypeptide is derived from the medicinal material, and identification of sika deer, red deer, dama dama and roe deer is realized.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a characteristic polypeptide, reagent kit, and identification method for distinguishing and identifying deer antler medicinal materials based on chemical markers. Background Technology

[0002] Deer antler is the main raw material for producing deer antler glue. The juice obtained by boiling and concentrating deer antlers solidifies into deer antler glue, while the by-product after boiling and air-drying is deer antler powder. Uncured young antlers—deer velvet—are more widely used and expensive in China. Manufacturers have a tradition of harvesting deer velvet, leading to a scarcity of deer antler resources in China, with most deer antlers being imported. Unlike the captive farming methods in China, deer in Eastern Europe are mostly free-range, resulting in a diverse range of deer breeds and crossbreeding. This makes identification difficult once the antlers are in the market, as they cannot be identified solely by their appearance.

[0003] In the 2020 edition of the Chinese Pharmacopoeia, deer antlers are defined as those of the red deer (Cervidae). Cervus elaphus Linnaeus ) or sika deer ( Cervus Nippon Temminck The ossified antlers or the antler bases that detach the following spring after velvet antlers are commonly referred to as "red deer antlers," "sika deer antlers," and "antler detachment," respectively. Currently, the quality of deer antlers is controlled solely by the "extractives" item, resulting in a single testing item and a lack of specific quality control indicators. Given these current conditions and problems, distinguishing and identifying deer antler medicinal materials is the primary task of quality control. Summary of the Invention

[0004] To address the technological gaps in the existing technology, this invention provides a characteristic polypeptide group for distinguishing and identifying medicinal materials and preparations of sika deer, red deer, fallow deer, and roe deer based on chemical markers.

[0005] The present invention also discloses a kit containing the above-mentioned characteristic polypeptide group for rapid detection and identification of deer-derived medicinal materials and their preparations.

[0006] Another objective of this invention is to disclose a method for distinguishing and identifying deer-derived medicinal materials and their preparations using the aforementioned characteristic polypeptide groups.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a characteristic polypeptide group for distinguishing and identifying deer-derived medicinal materials and their preparations based on chemical markers, specifically: VGGNAPAFGAEALER, VAEIGAEALGR, and VNVAEIGAEALGR. The present invention further discloses a kit containing the above-mentioned characteristic polypeptide group.

[0008] This invention provides a method for distinguishing and identifying deer-derived medicinal materials and their preparations based on chemical markers, specifically including the following steps: (1) Sample preparation: weigh the antler medicinal material powder, add denaturing buffer and DTT solution, extract by water bath shaking, cool down and centrifuge, filter the supernatant; filter the filtrate, add IAA solution, centrifuge after avoiding light reaction; mix the supernatant and water, centrifuge after desalination, collect the supernatant and add water again, repeat centrifugation once to prepare sample stock solution; (2) Mix the supernatant and water, add trypsin, homogenize by blowing and then enzymolysis, inactivate after reaction, obtain the sample solution for detection.

[0009] (3) The sample solution for detection is identified by high performance liquid chromatography-triple quadrupole mass spectrometry, and the species identification of antler medicinal material is realized according to whether the characteristic polypeptide peaks.

[0010] Further, in step (1), the specific process of sample preparation is as follows: weigh 40 mg of antler powder, add 2 mL of denaturing buffer and 200 μL of DTT solution, extract by 90℃ water bath shaking for 4 h, cool down and centrifuge, filter the supernatant with 0.22 μm filter head, filter 500 μL of filtrate, add 100 μL of IAA solution, centrifuge after avoiding light reaction for 30 min, mix equal volume of supernatant and water, centrifuge in 3 kDa ultrafiltration centrifuge tube to desalt, add water to the supernatant and repeat the operation once to prepare sample stock solution.

[0011] Further, in step (1), the denaturing buffer is 6M guanidine hydrochloride, 1M Tris, 2.5mM ethylenediaminetetraacetic acid; the concentration of DTT solution is 0.5M; the concentration of IAA solution is 0.55M.

[0012] Further, in step (2), the specific operation is as follows: mix 100 μL of supernatant and 100 μL of water, add 10 mg / ml of trypsin 10 μL, homogenize by blowing, and then enzymolysis at 37℃ for 4 h, inactivate at 100℃ for 5 min after reaction.

[0013] Further, in step (3), the conditions of high performance liquid chromatography are as follows: the chromatographic column is Agilent SB C18RRHD, 2.1 mm×100 mm, 1.7 μm; the column temperature is 43 ℃, the flow rate is 0.3 mL / min, the mobile phase A is 0.1% formic acid solution, the mobile phase B is acetonitrile solution, gradient elution is carried out, the injection volume is 5 μL. The mass spectrometry conditions are as follows: The gradient elution conditions are as follows: 0 min to 7 min, 8%B → 12%B; 7 min to 11 min, 12%B → 22%B; 11 min to 18 min, 22%B → 40%B; 18 min to 23 min, 40%B → 90%B; 23 min to 23.5 min, 90%B → 8%B; 23.5 min to 28 min, 8%B.

[0014] Furthermore, the mass spectrometry conditions are as follows: a triple quadrupole mass spectrometer detector, an electrospray ionization (ESI) source, positive ion mode, and multiple reaction monitoring are performed; the ion source spray gas flow rate is 40 μL / min; the auxiliary heating gas flow rate is 40 μL / min; the ionization voltage is 5.5 kV; the ion source temperature is 550 ℃; the cone voltage is 47 V; and the collision voltage is 80 V.

[0015] Furthermore, in step (3), the specific identification principle is as follows: when the characteristic peak of VGGNAPAFGAEALER appears, it is considered to be the antler of a sika deer or a red deer; when the characteristic peak of VAEIGAEALGR appears, it is considered to be the antler of a roe deer; when the characteristic peak of VNVAEIGAEALGR appears, it is considered to be the antler of a fallow deer.

[0016] The chemical markers used in this invention are chemical substances unique to a particular medicinal material (processed slices) rather than originating from other medicinal materials, which are used as identification indicators for the authentication and quality control of traditional Chinese medicine. This invention uses characteristic polypeptides as chemical markers for deer antler medicinal materials, enabling the differentiation and identification of multiple species.

[0017] The chemical markers used in this invention—characteristic polypeptides—are all derived from the medicinal materials themselves, and they enable the identification of sika deer, red deer, fallow deer, and roe deer.

[0018] The beneficial effects of this invention are as follows: (1) The identification technique of the present invention has a highly efficient sample processing method and complete sample lysis; the instruments and database used have high sensitivity and the data analysis results are reliable.

[0019] (2) Based on the concept of chemical markers, this invention combines proteomics and peptideomics to develop a detection method that can be used to identify red deer, sika deer, fallow deer and roe deer, laying the foundation for filling the gaps in the current standards. Attached Figure Description

[0020] Figure 1 The total ion chromatogram of characteristic polypeptides from red deer; Figure 2 The total ion chromatogram of characteristic polypeptides from sika deer; Figure 3 The total ion chromatogram of characteristic polypeptides from fallow deer; Figure 4 Total ion chromatogram of characteristic polypeptides from roe deer; Figure 5 This represents the number of proteins and peptides obtained from the database search. Figure 6 Comparison of the number of differentially expressed polypeptides among different deer species; Figure 7 The results are for validation of peptide 1 sequence; Figure 8 The results are for validation of peptide 2 sequence; Figure 9 The results are for validation of peptide 3 sequence; Figure 10 The specificity results of polypeptides for red deer or sika deer specific characteristics; Figure 11 Results of polypeptide specificity for roe deer; Figure 12 Results of polypeptide specificity for fallow deer; Figure 13 The sequence VGGNAPAFGAEALER b and y ion assignments and spiking verification results are presented. Figure 14 The results of sequence VAEIGAEALGR b and y ion assignment and spiking verification; Figure 15 The sequence VNVAEIGAEALGR b and y ion assignments and spiking verification results are presented. Detailed Implementation

[0021] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0022] The instruments and materials used in the embodiments provided by this invention are as follows: (1) Materials Guanidine hydrochloride, Tris(hydroxymethyl)aminomethane (Tris), EDTA, DTT, Iodoacetamide (IAA), ammonium bicarbonate, and acetic acid were all of analytical grade. Distilled water; formic acid and acetonitrile were of spectroscopic grade; trypsin (Sigma, lot number: T8802).

[0023] (2) Instruments EASY-nLC 1000 nanoliter liquid chromatography, Scientific OrbitrapFusion high-resolution mass spectrometry (Thermo Fisher Scientific, USA); Triple Quad 6500+ high performance liquid chromatography-mass spectrometry system (SCIEX Corporation, USA); XSE205 electronic balance (Sartorius GmbH, Germany).

[0024] Sample preparation: The antler sample was chopped into a fine powder. 40 mg of the powder was accurately weighed and added to 2 mL of denaturing buffer (6 mol / L guanidine hydrochloride, 1 mol / L Tris, 2.5 mmol / L ethylenediaminetetraacetic acid) and 200 μL of DTT solution (0.5 mol / L). The mixture was extracted by shaking in a 90 ℃ water bath for 4 h. After cooling, the mixture was centrifuged, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter. 500 μL of the filtrate was collected, and 100 μL of IAA solution (0.55 mol / L) was added. The mixture was reacted in the dark for 30 min, centrifuged, and 500 μL of the supernatant was collected and mixed with 500 μL of water. The mixture was then centrifuged at 10000 r for 10 min in a 3 kDa ultrafiltration centrifuge tube to remove salts. The supernatant was collected, and 500 μL of water was added. The centrifugation was repeated once more to obtain the sample stock solution. Take 100 μL of supernatant and 100 μL of water, mix well, add 10 μL of bovine trypsin (10 mg / mL, freshly prepared before use), mix well by pipetting, and incubate at 37 ℃ for 4 h. After the reaction is complete, inactivate at 100 ℃ for 5 min and set aside for later use.

[0025] Example 1 This embodiment involves collecting total ion current data of antler polypeptides, and the specific steps are as follows: Nano-level liquid chromatography-high-resolution mass spectrometry (NLC-HDMS) was used to detect antler peptides. NLC conditions: Desalting and enrichment were performed using a Thermo Acclaim PepMap C18 column (100 μm × 3.5 cm, 5 μm), followed by separation using a Thermo Acclaim PepMap C18 column (75 μm × 15 cm, 3 μm). The flow rate was 300 nL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% acetonitrile in 0.1% formic acid aqueous solution. Gradient elution was performed (0–1 min, 1% B → 6% B; 1–96 min, 6% B → 22% B; 96–113 min, 22% B → 30% B; 113–117 min, 30% B → 95% B; 117–120 min, 95% B). The injection volume was 1 μL.

[0026] High-resolution mass spectrometry conditions: The ion source was a Nanospray Flex nano-spray source in positive ion mode, with a spray voltage of 2200 V, an ion transport capillary temperature of 275 ℃, and an S-Lens transmission efficiency of 60%. An Orbitrap mass analyzer was used, with a first-stage mass spectrometry resolution of 60,000 m / s and an acquisition range of 350–1550 m / z. Second-stage mass spectrometry employed Rapid Scan mode, using Top 20 data-dependent selection for precursor ion selection, and HCD mode for fragmentation.

[0027] Figures 1-4 The total ion chromatograms of different deer species show that there are certain differences among the species.

[0028] Example 2 This embodiment describes the screening of peptides with potential species-specific characteristics, and the specific steps are as follows: The data obtained in Example 1 were combined with the database for chemometric analysis of potential specific peptides. Specificity verification of the antler chemical markers was performed using high-performance liquid chromatography-triple quadrupole mass spectrometry (MRM) mode. Liquid chromatography conditions: Agilent SB C18 RRHD column (2.1 mm × 100 mm, 1.7 μm), column temperature 43 ℃, flow rate 0.3 mL / min, mobile phase A was 0.1% (v / v) formic acid solution, and mobile phase B was acetonitrile solution. Gradient elution was performed (0 min–7 min, 8% B → 12% B; 7 min–11 min, 12% B → 22% B; 11 min–18 min, 22% B → 40% B; 18 min–23 min, 40% B → 90% B; 23 min–23.5 min, 90% B → 8% B; 23.5 min–28 min, 8% B), with an injection volume of 5 μL. Mass spectrometry conditions: A triple quadrupole mass spectrometer detector, electrospray ionization (ESI) source, positive ion mode, and multiple reaction monitoring were used; ion source spray gas flow rate: 40 μL / min; auxiliary heating gas flow rate: 40 μL / min; ionization voltage: 5.5 kV; ion source temperature: 550 °C; cone voltage: 47 V; collision voltage: 80 V. If a specific ion peak is detected only in a sample of a particular species, that ion pair is determined to be a characteristic polypeptide ion pair of that species. Figure 5 and Figure 6 As shown.

[0029] The specific characteristic polypeptide sequences and mass spectrometry information of antler are shown in Table 1.

[0030] Table 1 Example 3 This embodiment confirms the sequence of a specific characteristic polypeptide, and the specific steps are as follows: Species-representative peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and the ions corresponding to the peptide sequences were spiked and verified using high-performance liquid chromatography-triple quadrupole mass spectrometry (MRM mode). Verification was performed according to the liquid chromatography and mass spectrometry conditions described in Example 2. The mass spectrometric amino acid sequences of peptides 1, 2, and 3 are as follows: Figures 7-9As shown. Samples from sika deer, red deer, fallow deer, and roe deer were processed according to the sample preparation method, and then the specificity of their sequences was verified using triple quadrupole liquid chromatography-mass spectrometry (LC-MS / MS). Figures 10-12 As shown.

[0031] Example 1: Confirmation of the characteristic polypeptide amino acid sequence Each characteristic peptide was artificially synthesized according to its amino acid sequence as a reference. The peptide sequences were confirmed using HPLC-QQQ MS in MRM mode, based on de novo sequencing results and specificity results obtained using Peaks Studio software. The breakpoints of each characteristic peptide were extracted individually for mass spectrometry analysis to determine sequence accuracy. The accuracy of the peptide sequence was inferred by comparing the retention times of the synthesized peptide reference, sample, and spiked sample in the mass spectra. The secondary mass spectra and the assignment and verification results of b and y ions are shown below. Figures 13-15 .

[0032] By extracting secondary γ ions from each sequence for fragmentation site verification, the retention times of each γ ion in the mass spectra of the synthetic reference solution, sample solution, and spiked sample solution were consistent. The experimental results confirmed that the sequence of peptide 1 was VGGNAPAFGAEALER, the sequence of peptide 2 was VAEIGAEALGR, and the sequence of peptide 3 was VNVAEIGAEALGR.

[0033] Example 2 Preparation of bovine trypsin solution: Take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to prepare a solution containing 10 mg per 1 mL, and prepare it immediately before use.

[0034] Preparation of the test solution: The antler sample was chopped into a fine powder. 40 mg of the sample powder was accurately weighed and added to 2 mL of denaturing buffer (6 mol / L guanidine hydrochloride, 1 mol / L Tris, 2.5 mmol / L ethylenediaminetetraacetic acid) and 200 μL of DTT solution (0.5 mol / L). The mixture was extracted by shaking in a water bath at 90℃ for 4 h. After cooling, the mixture was centrifuged, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter. 500 μL of the filtrate was collected, and 100 μL of IAA solution (0.55 mol / L) was added. The mixture was reacted in the dark for 30 min, centrifuged, and 500 μL of the supernatant was collected and mixed with 500 μL of water. The mixture was then centrifuged at 10000 r for 10 min in a 3 kDa ultrafiltration centrifuge tube to remove salts. The supernatant was collected, and 500 μL of water was added. The centrifugation was repeated once to obtain the sample stock solution. Take 100 μL of supernatant and 100 μL of water, mix well, add 10 μL of bovine trypsin (10 mg / mL, freshly prepared before use), mix well by pipetting, and incubate at 37 ℃ for 4 h. After the reaction is complete, inactivate at 100 ℃ for 5 min and set aside for later use.

[0035] Fifty-one batches of DNA-sequencing samples were prepared into test solutions according to the sample preparation method described above. Ion pairs were selected as follows: ① mass-to-charge ratio m / z 729.9 (double charge) → 892.5 and m / z 729.9 (double charge) → 1060.6; ② 543.3 (double charge) → 416.3 and m / z 543.3 (double charge) → 673.4; ③ 649.9 (double charge) → 786.5 and m / z 649.9 (double charge) → 916.5. These were then analyzed. Specific results are shown in Table 2.

[0036] Table 2

Claims

1. A characteristic polypeptide group for distinguishing and identifying deer-derived medicinal materials and their preparations based on chemical markers, characterized in that, The characteristic polypeptide group is: VGGNAPAFGAEALER, VAEIGAEALGR, VNVAEIGAEALGR.

2. A kit containing the characteristic polypeptide group of claim 1.

3. A method for distinguishing and identifying deer-derived medicinal materials and their preparations based on the characteristic polypeptide group described in claim 1, characterized in that, Includes the following steps: (1) Sample preparation: Weigh the deer antler powder, add denaturing buffer and DTT solution, extract by shaking in a water bath, centrifuge after cooling, and filter the supernatant; absorb the filtrate, add IAA solution, react in the dark and centrifuge; absorb the supernatant and mix with water, desalt and centrifuge, collect the supernatant and add water again, repeat centrifugation once to obtain the sample stock solution; (2) Take the supernatant and water, add trypsin, mix well and then enzymatically digest. After the reaction is complete, inactivate the enzyme and obtain the sample solution to be tested for later use. (3) The sample solution to be tested is identified by high performance liquid chromatography-triple quadrupole mass spectrometry. The authenticity of the species of deer antler medicinal material is determined by whether the characteristic polypeptides elute.

4. The method according to claim 3, characterized in that, In step (1), the specific process of sample preparation is as follows: Weigh 40 mg of deer antler powder, add 2 mL of denaturing buffer and 200 μL of DTT solution, shake and extract in a 90℃ water bath for 4 h, cool and centrifuge, take the supernatant and filter it through a 0.22 μm filter, take 500 μL of filtrate, add 100 μL of IAA solution, react in the dark for 30 min, centrifuge, take an equal volume of supernatant and water and place them in a 3 kDa ultrafiltration centrifuge tube for centrifugation to remove salt, add water to the supernatant and repeat the operation once to obtain the sample stock solution.

5. The method according to claim 3, characterized in that, In step (1), the denaturing buffer is 6M guanidine hydrochloride, 1M Tris, and 2.5 mM ethylenediaminetetraacetic acid; the concentration of the DTT solution is 0.5 M; and the concentration of the IAA solution is 0.55 M.

6. The method according to any one of claims 3-5, characterized in that, In step (2), the specific operation is as follows: after mixing 100 μL of supernatant and 100 μL of water, add 10 μL of 10 mg / ml trypsin, mix well and then enzymatically hydrolyze at 37℃ for 4 h. After the reaction is completed, inactivate at 100℃ for 5 min.

7. The method according to claim 3, characterized in that, In step (3), the conditions for high performance liquid chromatography are: the chromatographic column is an Agilent SB C18 RRHD, 2.1 mm × 100 mm, 1.7 μm; The column temperature was 43 ℃, the flow rate was 0.3 mL / min, the mobile phase A was 0.1% formic acid solution and B was acetonitrile solution, gradient elution was performed, and the injection volume was 5 μL.

8. The method according to claim 5, characterized in that, The gradient elution conditions are as follows: 0 min to 7 min, 8%B → 12%B; 7 min to 11 min, 12%B → 22%B; 11 min to 18 min, 22%B → 40%B; 18 min to 23 min, 40%B → 90%B. 23 min~23.5 min, 90%B→8%B, 23.5 min~28 min, 8%B.

9. The method according to claim 1, 5, or 6, characterized in that, The mass spectrometry conditions were as follows: a triple quadrupole mass spectrometer detector, an electrospray ionization source, positive ion mode, and multiple reaction monitoring were performed; the ion source spray gas flow rate was 40 μL / min; the auxiliary heating gas flow rate was 40 μL / min; the ionization voltage was 5.5 kV; the ion source temperature was 550 ℃; the cone voltage was 47 V; and the collision voltage was 80 V.

10. The method according to any one of claims 3-9, characterized in that, In step (3), the specific identification principle is as follows: when the characteristic peak of VGGNAPAFGAEALER appears, it is considered to be the antler of a sika deer or a red deer; when the characteristic peak of VAEIGAEALGR appears, it is considered to be the antler of a roe deer; when the characteristic peak of VNVAEIGAEALGR appears, it is considered to be the antler of a fallow deer.

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