Deer-derived specific peptide and detection method therefor

Abstract

Disclosed are a deer-derived specific peptide and a detection method therefor; by screening through a large number of experiments, a ratio of relative contents of two deer-derived peptides is determined, and a graph is drawn by using a proportion of a deer antler gelatin in a mixed gelatin as an abscissa and using a value of A.sub.peptide 1/A.sub.peptide 2 as an ordinate; the proportion of the deer antler gelatin is linear with A.sub.peptide 1/A.sub.peptide 2 as a standard curve equation to distinguish a deer hide gelatin from the deer antler gelatin; the method can be used for distinguishing the deer antler gelatin from the deer hide gelatin, and controlling the quality; a defect in the prior art that the deer antler gelatin and the deer hide gelatin are difficult to distinguish in appearance, and are also difficult to distinguish by using a specific peptide fragment, is solved.

Claims

1. A detection method for a specific peptide capable of distinguishing a deer antler gelatin from a deer hide gelatin, comprising the following steps of: (i) preparing two deer-derived characteristic peptides, peptide 1 and peptide 2, dissolving the peptide 1 and the peptide 2 in water to yield a mixed control solution; and (ii) digesting a deer gelatin sample from Cervus nippon Temminck or Cervus elaphus Linnaeus with trypsin to yield an enzymatic hydrolysate, then injecting the enzymatic hydrolysate and the mixed control solution into a liquid chromatograph/mass spectrometer, taking the peptide 1 and the peptide 2 as controls, and adopting an ESI positive ion mode and a multi-reaction monitoring mode for detection, wherein selected ion pairs comprise: the peptide 1: m/z 850.4 triple charge.fwdarw.515.4, the peptide 2: m/z 845.0 triple charge.fwdarw.507.3; and determining whether the deer gelatin sample is the deer hide gelatin derived from Cervus elaphus Linnaeus or the deer antler gelatin derived from Cervus nippon Temminck by a ratio of a peak area A.sub.peptide 1 of the peptide 1 to a peak area A.sub.peptide 2 of the peptide 2; A.sub.peptide 1/A.sub.peptide 2 of the deer antler gelatin is higher than 5.5, while A.sub.peptide 1/A.sub.peptide 2 of the deer hide gelatin is lower than 1.2; wherein the peptide 1 has the amino acid sequence shown as SEQ ID NO: 01; and the peptide 2 has the amino acid sequence shown as SEQ ID NO: 02; liquid phase conditions for detection by the liquid chromatograph/mass spectrometer are as follows: a chromatographic column is a 1.7 m HPLC column with a specification of 2.1 m100 mm, a sample size of 2 l and a flow rate of 0.3 ml/min; 10% to 30% A linear gradient elution lasts for 0 to 3.5 minutes, 30% to 10% A linear gradient elution lasts for 3.5 minutes to 4 minutes, and 10% A linear gradient elution lasts for 4 minutes to 6 minutes.

2. The detection method according to claim 1, wherein a method for the digesting the deer gelatin sample comprises: adding 5 ml of phosphate buffer solution into 10 mg of the deer gelatin sample, completely dissolving the sample by ultrasound to yield a solution, then centrifuging the solution at 12,000 rpm for 20 minutes, taking and transferring 150 l of supernatant from the centrifuged solution into a 2 ml centrifuge tube, diluting the supernatant with 1 ml of 50 mM PBS, adding the trypsin to yield a mixture, shaking the mixture evenly for an enzymolysis, adding 60 l of 10% v/v trifluoroacetic acid solution to stop the mixture, centrifuging the mixture at 12,000 rpm for 20 minutes to obtain the enzymatic hydrolysate.

3. The detection method according to claim 2, wherein an amount of the trypsin is from 0.1 wt % to 10 wt %.

4. The detection method according to claim 2, wherein the enzymolysis is selected from the group consisting of constant-temperature enzymolysis at 37 C., microwave-assisted enzymolysis, ultrasonic-assisted enzymolysis and enzyme-immobilized enzymolysis.

5. A detection method for a specific peptide capable of distinguishing a deer antler gelatin from a deer hide gelatin, comprising the following steps of: (i) Establishment of Linear Relationship mixing the deer antler gelatin and the deer hide gelatin according to proportions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% respectively, adding 5 ml of phosphate buffer solution into 10 mg of mixed gelatin sample in each batch, completely dissolving the sample by ultrasound to yield a solution, centrifuging the solution at 12,000 rpm for 20 minutes, taking and transferring 150 l of supernatant from the centrifuged solution into a 2 ml centrifuge tube, diluting the supernatant with 1 ml of 50 mM PBS, adding trypsin with a mass concentration of 1% to yield a mixture, shaking the mixture evenly for microwave enzymolysis for 30 minutes, adding 60 l of 10% trifluoroacetic acid solution to stop the enzymolysis to yield an enzymolysis solution, centrifuging the enzymolysis solution at 12,000 rpm for 20 minutes to obtain enzymatic solutions of mixed gelatin samples with different proportions; and injecting the enzymatic hydrolysates of the mixed gelatin samples with different proportions into a liquid chromatograph/mass spectrometer, a sample size is 1 g, and liquid phase conditions for detection by the liquid chromatograph/mass spectrometer are as follows: a chromatographic column is a 1.7 m C.sub.18 reversed phase column with a specification of 2.1 m100 mm and a flow rate of 0.3 ml/min, a mobile phase A is acetonitrile, a mobile phase B is 0.1% formic acid, 10% to 30% A linear gradient elution lasts for 0 to 3.5 minutes, 30% to 10% A linear gradient elution lasts for 3.5 minutes to 4 minutes, and 10% A elution lasts for 4 minutes to 6 minutes; and a mass spectrometry condition for detection by the liquid chromatograph/mass spectrometer is: an electrospray positive ion mode ESI+, and mass spectrometry parameters comprise: an ion source temperature of 500 C.; an ionization voltage of 5,500 V; a desolvent temperature of 500 C.; an ion source gas 1 of 60 psi; and an ion source gas 2 of 60 psi; setting the ion pair conditions corresponding to specific peptides as follows: peptide 1: M/z 850.4 triple charge.fwdarw.515.4, DP=162.49, CE=32.87; and peptide 2: m/z 845.0 triple charge.fwdarw.507.3, DP=162.52, CE=31.51; and drawing a graph by using a proportion of the deer antler gelatin in the mixed gelatin as an abscissa and using a value of A.sub.peptide 1/A.sub.peptide 2 as an ordinate, and since the proportion of the deer antler gelatin has a linear relationship to A.sub.peptide 1/A.sub.peptide 2, establishing a standard curve equation as: y=4.7903x+0.4106, and R.sup.2=0.9669; and since the proportion of the deer antler gelatin adulterated has a linear relationship with peak values of the peptide 1 and the peptide 2, calculating a ratio of A.sub.peptide 1/A.sub.peptide 2 according to the standard curve equation, and determining a mixing proportion of the deer antler gelatin and the deer hide gelatin; A.sub.peptide 1/A.sub.peptide 2 of the deer antler gelatin is equal to or higher than 5.5, while A.sub.peptide 1/A.sub.peptide 2 of the deer hide gelatin is equal to or lower than 1.2; the peptide 1 has the amino acid sequence shown as SEQ ID NO: 1; and the peptide 2 has the amino acid sequence shown as SEQ ID NO: 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a numerical relationship between a proportion of a deer antler gelatin in a mixed gelatin and A.sub.peptide 1/A.sub.peptide 2;

(2) FIG. 2 is a mass spectrogram of the peptide 1; and

(3) FIG. 3 is a mass spectrogram of the peptide 2.

DETAILED DESCRIPTION

(4) The present invention will be further described in detail hereinafter with reference to the specific embodiments, but the present invention is not limited to these embodiments.

(5) The trypsin used in the following embodiments was purchased from Promega Company.

Embodiment 1

(6) A deer-derived specific peptide had two specific peptide sequences, as shown in sequence table 1: peptide 1: Gly-Asn-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Hyp-Gly-Ala-Val-Gly-Ala-Lys; and peptide 2: Gly-Asn-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.

(7) The polypeptides above were prepared by Nanjing GenScript Biotech Corporation using a solid phase synthesis method.

(8) Embodiment 2 Proportional relationship between peptide 1 and peptide 2 of deer hide gelatin

(9) batches of commercially available deer hide gelatin samples were taken, with each batch of about 10 mg, added with 5 ml of phosphate buffer solution (pH=7.8), the samples were completely dissolved by ultrasound, centrifuged at 12,000 rpm for 20 minutes, 150 l of supernatant were placed into a 2 ml centrifuge tube, diluted with 1 ml of 50 mM PBS, added with 1 wt % trypsin, shaken evenly, and enzymolyzed at a constant temperature of 37 C. for 12 hours. After enzymolysis, 60 l of 10% v/v TFA solution was added to stop the reaction, and then centrifuged at 12,000 rpm for 20 minutes to obtain the enzymatic hydrolysate of the deer hide gelatin medical material, which was stored at 20 C. for later use.

(10) The enzymatic hydrolysates of each batch of deer hide gelatin were injected into the liquid chromatograph/mass spectrometer for detection, wherein a sample size was 1 g, and liquid phase conditions for detection by the liquid chromatograph/mass spectrometer were as follows: a chromatographic column was a 1.7 m C18 reversed phase column (2.1 m100 mm) with a flow rate of 0.3 ml/min, a mobile phase A was acetonitrile, a mobile phase B was 0.1% formic acid, 10% to 30% A linear gradient elution lasted for 0 to 3.5 minutes, 30% to 10% A linear gradient elution lasts for 3.5 minutes to 4 minutes, and 10% A elution lasts for 4 minutes to 6 minutes. A mass spectrometry condition for detection by the liquid chromatograph/mass spectrometer was: an electrospray positive ion mode ESI+, and mass spectrometry parameters comprised: an ion source temperature of 500 C.; an ionization voltage of 5,500 V; a desolvent temperature of 500 C.; an ion source gas 1 of 60 psi; and an ion source gas 2 of 60 psi. The mass spectra were shown in FIG. 2 and FIG. 3. The ion pair conditions corresponding to the specific peptides were set as follows: peptide 1: M/z 850.4 (triple charge).fwdarw.515.4, DP=162.49, CE=32.87; and peptide 2: m/z 845.0 (triple charge).fwdarw.507.3, DP=162.52, CE=31.51.

(11) The values of A.sub.peptide 1/A.sub.peptide 2 in the 10 batches of deer hide gelatin were shown in Table 1. The average value of A.sub.peptide 1/A.sub.peptide 2 was 0.0.6120.282.

(12) TABLE-US-00001 TABLE 1 Results of A.sub.peptide 1/A.sub.peptide 2 in deer hide gelatin Average value A.sub.peptide 1/ of A.sub.peptide 1/ Batch A.sub.peptide 1 A.sub.peptide 2 A.sub.peptide 2 A.sub.peptide 2 Deer hide 117972 285071 0.414 0.612 0.282 gelatin-1 Deer hide 94534 197073 0.480 gelatin-2 Deer hide 147007 335823 0.438 gelatin-3 Deer hide 93660 88906 1.053 gelatin-4 Deer hide 124893 146508 0.852 gelatin-5 Deer hide 103489 282973 0.366 gelatin-6 Deer hide 128678 115750 1.112 gelatin-7 Deer hide 73454 181928 0.404 gelatin-8 Deer hide 77833 155640 0.500 gelatin-9 Deer hide 99419 197361 0.504 gelatin-10

(13) Embodiment 3 Proportional relationship between peptide 1 and peptide 2 of deer antler gelatin

(14) batches of deer antler samples were taken and prepared into deer antler gelatin samples according to the method of preparing deer antler gelatin in Chinese Pharmacopoeia 2020, with each batch of 10 mg, added with 5 ml of phosphate buffer solution (pH=7.8), the samples were completely dissolved by ultrasound, centrifuged at 12,000 rpm for 20 minutes, 150 l of supernatant were placed into a 2 ml centrifuge tube, diluted with 1 ml of 50 mM PBS, added with 1 wt % trypsin, shaken evenly, and enzymolyzed by ultrasound for 10 minutes. After enzymolysis, 60 l of 10% v/v TFA solution was added to stop the reaction, and then centrifuged at 12,000 rpm for 20 minutes to obtain the enzymatic hydrolysate of the deer antler gelatin, which was stored at 20 C. for later use.

(15) The enzymatic hydrolysates of each batch of deer antler gelatin were injected into the liquid chromatograph/mass spectrometer for detection, wherein a sample size was 1 g, and liquid phase conditions for detection by the liquid chromatograph/mass spectrometer were as follows: a chromatographic column was a 1.7 m C18 reversed phase column (2.1 m100 mm) with a flow rate of 0.3 ml/min, a mobile phase A was acetonitrile, a mobile phase B was 0.1% formic acid, 10% to 30% A linear gradient elution lasted for 0 to 3.5 minutes, 30% to 10% A linear gradient elution lasted for 3.5 minutes to 4 minutes, and 10% A elution lasted for 4 minutes to 6 minutes. A mass spectrometry condition for detection by the liquid chromatograph/mass spectrometer was: an electrospray positive ion mode ESI+, and mass spectrometry parameters comprised: an ion source temperature of 500 C.; an ionization voltage of 5,500 V; a desolvent temperature of 500 C.; an ion source gas 1 of 60 psi; and an ion source gas 2 of 60 psi. The mass spectra were shown in FIG. 2 and FIG. 3. The ion pair conditions corresponding to the specific peptides were set as follows: peptide 1: M/z 850.4 (triple charge).fwdarw.515.4, DP=162.49, CE=32.87; and peptide 2: m/z 845.0 (triple charge).fwdarw.507.3, DP=162.52, CE=31.51.

(16) The values of A.sub.peptide 1/A.sub.peptide 2 in the 10 batches of deer antler gelatin were shown in Table 2. The average value of A.sub.peptide 1/A.sub.peptide 2 was 7.4281.617.

(17) TABLE-US-00002 TABLE 2 Results of A.sub.peptide 1/A.sub.peptide 2 in deer antler gelatin Average value A.sub.peptide 1/ of A.sub.peptide 1/ Batch A.sub.peptide 1 A.sub.peptide 2 A.sub.peptide 2 A.sub.peptide 2 Deer antler 1733347 238417 7.270 7.428 1.617 gelatin-1 Deer antler 1874077 205064 9.139 gelatin-2 Deer antler 1736568 289973 5.989 gelatin-3 Deer antler 1217432 182921 6.656 gelatin-4 Deer antler 1739091 289409 6.009 gelatin-5 Deer antler 1859076 246296 7.548 gelatin-6 Deer antler 1802154 186993 9.638 gelatin-7 Deer antler 1945171 311810 6.238 gelatin-8 Deer antler 1758124 175207 10.035 gelatin-9 Deer antler 1418918 246354 5.760 gelatin-10

(18) Embodiment 4 Proportional relationships between peptide 1 and peptide 2 in mixed samples of deer hide gelatin and deer antler gelatin with different proportions

(19) The deer antler gelatin and the deer hide gelatin were mixed according to proportions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, respectively added with 5 ml of phosphate buffer solution (pH-7.8) into about 10 mg of mixed gelatin sample in each batch, the sample was completely dissolved by ultrasound, centrifuged at 12,000 rpm for 20 minutes, 150 l of supernatant was placed into a 2 ml centrifuge tube, diluted with 1 ml of 50 mM PBS, added with 1 wt % trypsin, shaken evenly for microwave enzymolysis for 30 minutes, added with 60 l of 10% v/v TFA solution to stop the reaction after enzymolysis, centrifuged at 12,000 rpm for 20 minutes to obtain enzymolysis solutions of mixed gelatin samples with different proportions, which were stored at 20 C. for later use.

(20) The enzymatic hydrolysates of the mixed gelatin samples with different proportions were injected into the liquid chromatograph/mass spectrometer, wherein a sample size was 1 g, and liquid phase conditions for detection by the liquid chromatograph/mass spectrometer are as follows: a chromatographic column was a 1.7 m C18 reversed phase column (2.1 m100 mm) with a flow rate of 0.3 ml/min, a mobile phase A was acetonitrile, a mobile phase B was 0.1% formic acid, 10% to 30% A linear gradient elution lasted for 0 to 3.5 minutes, 30% to 10% A linear gradient elution lasted for 3.5 minutes to 4 minutes, and 10% A elution lasted for 4 minutes to 6 minutes. A mass spectrometry condition for detection by the liquid chromatograph/mass spectrometer was: an electrospray positive ion mode ESI+, and mass spectrometry parameters comprised: an ion source temperature of 500 C.; an ionization voltage of 5,500 V; a desolvent temperature of 500 C.; an ion source gas 1 of 60 psi; and an ion source gas 2 of 60 psi. The mass spectra were shown in FIG. 2 and FIG. 3. The ion pair conditions corresponding to the specific peptides were set as follows: peptide 1: M/z 850.4 (triple charge).fwdarw.515.4, DP=162.49, CE=32.87; and peptide 2: m/z 845.0 (triple charge).fwdarw.507.3, DP=162.52, CE=31.51.

(21) Values of A.sub.peptide 1/A.sub.peptide 2 of mixed gelatin samples with different proportions were shown in Table 3, and a numerical relationship between the proportion of the deer antler gelatin in the mixed gelatin and A.sub.peptide 1/A.sub.peptide 2 was shown in FIG. 1. A graph was drawn by using the proportion of the deer antler gelatin in the mixed gelatin as an abscissa and using the value of A.sub.peptide 1/A.sub.peptide 2 as an ordinate, and the proportion of the deer antler gelatin had a linear relationship to A.sub.peptide 1/A.sub.peptide 2, y=4.7903x+0.4106, and R.sup.2=0.9669. It was indicated that the proportion of the deer antler gelatin adulterated was related to the peak areas of the peptide 1 and the peptide 2. Therefore, the mixing proportion of the deer antler gelatin and the deer hide gelatin could be determined according to the ratio of A.sub.peptide 1/A.sub.peptide 2.

(22) TABLE-US-00003 TABLE 3 Relationship between the mixing proportion of the deer antler gelatin/deer hide gelatin mixed sample and the peak area Mixed gelatin proportion Proportion Proportion Peak area of deer of deer ratio antler hide Peak area A.sub.peptide 1/ gelatin % gelatin % Peptide 1 Peptide 2 A.sub.peptide 2 0 100 341004 415023 0.822 10 90 329057 370689 0.888 20 80 513434 387355 1.325 30 70 755737 419440 1.802 40 60 770995 385569 2.000 50 50 1076657 443720 2.426 60 40 1140066 361642 3.152 70 30 1449246 396946 3.651 80 20 1755511 359593 4.882 90 10 1710217 360363 4.746 100 0 2179560 421602 5.170

Embodiment 5

(23) Mixed control of the peptide 1 and the peptide 2 with a certain concentration were taken, and fed for six times continuously under the above-mentioned chromatography-mass spectrometry conditions, to determine the peak areas of the control of the peptide 1 and the peptide 2, and calculate the RSD of the peak areas of the control. The results were shown in Table 4, and the RSD of the peptide 1 and the peptide 2 were respectively 1.10% and 1.67%, indicating that the method had excellent precision.

(24) A corresponding concentration when a signal-to-noise ratio (S/N) of the peptide 1 and the peptide 2 was about 3, was taken as a limit of detection (LOD), and a corresponding concentration when the signal-to-noise ratio (S/N) of the peptide 1 and the peptide 2 was about 10, was taken as a limit of quantitation (LOQ). The results were shown in Table 4, and the LOQ and the LOD of the peptide 1 were 0.72 ng/ml and 0.24 ng/ml respectively. The LOQ and the LOD of the peptide 2 were 2.40 ng/ml and 0.80 ng/ml respectively.

(25) TABLE-US-00004 TABLE 4 Precision, limit of detection and limit of quantitation of peptide 1 and peptide 2 Precision LOQ (RSD, %) (ng/ml) LOD (ng/ml) Peptide 1 1.10 0.72 0.24 Peptide 2 1.67 2.40 0.80