Deer-derived specific peptide and detection method therefor

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 μl 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 μl 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 μg, and liquid phase conditions for detection by the liquid chromatograph/mass spectrometer are as follows:
• a chromatographic column is a 1.7 μm C18 reversed phase column with a specification of 2.1 μm×100 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→515.4, DP=162.49, CE=32.87;
• and
• peptide 2:
• m/z 845.0 triple charge→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 Apeptide 1/Apeptide 2 as an ordinate, and since the proportion of the deer antler gelatin has a linear relationship to Apeptide 1/Apeptide 2, establishing a standard curve equation as:
• y=4.7903x+0.4106, and R2=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 Apeptide 1/Apeptide 2 according to the standard curve equation, and determining a mixing proportion of the deer antler gelatin and the deer hide gelatin;
• Apeptide 1/Apeptide 2 of the deer antler gelatin is equal to or higher than 5.5, while Apeptide 1/Apeptide 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.