CN113004374B - Deer glue characteristic peptide and method for identifying deer glue contained in sample to be detected - Google Patents
Deer glue characteristic peptide and method for identifying deer glue contained in sample to be detected Download PDFInfo
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- CN113004374B CN113004374B CN202110422395.4A CN202110422395A CN113004374B CN 113004374 B CN113004374 B CN 113004374B CN 202110422395 A CN202110422395 A CN 202110422395A CN 113004374 B CN113004374 B CN 113004374B
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Abstract
The invention discloses a deer-horn glue characteristic peptide and a method for identifying whether a sample to be detected contains deer-horn glue. When the liquid chromatography-tandem mass spectrometry method is adopted to carry out multi-reaction detection in an electrospray negative ion mode, the parent ion of the deer-horn glue characteristic peptide is 732.84, and the daughter ions are 875.42 and 818.40; alternatively, the parent ion is 675.99 and the daughter ion is 914.46, 746.38. The deer-horn glue characteristic peptide can identify and identify deer-horn glue medicinal materials, and a high performance liquid chromatography-tandem mass spectrometry rapid detection method (HPLC-MS/MS) taking characteristic peptide fragment ion pairs as indexes can be established on the basis of the deer-horn glue characteristic peptide.
Description
The application is a divisional application with the application date of 2018, 9 and 3, the application number of CN201811021204.8, and the name of the invention is a deer glue characteristic peptide and a method for identifying whether a sample to be detected contains deer glue.
Technical Field
The invention belongs to the technical field of medicine and food detection, and particularly relates to a detection technology for identifying the truth of deer-horn gelatin and products thereof by using a high performance liquid chromatography-tandem mass spectrometry technology.
Background
Deer glue (also called as deer skin glue) is a special traditional Chinese medicine prepared by decocting and processing the skin of red deer Cervus elaphus Linnaeus or Cervus nippon Temminck, and has the effects of tonifying kidney, strengthening yang, replenishing blood and stopping bleeding. The compendium of materia medica records that the deerskin is nontoxic and treats all kinds of anal leakage, leucorrhea, metrorrhagia, kidney deficiency and spermatorrhea. Since ancient times, deer-horn glue is a functional health-care food for nourishing and keeping fit, and in recent years, with the improvement of living standard, deer-horn glue medicinal materials and products thereof are receiving more and more attention.
Because the deer-horn glue has obvious efficacy, high price and relatively short raw materials, the counterfeit deer-horn glue produced by illegally adopting other animal skin or mixed skin glue appears in the market, so that the quality of deer-horn glue medicinal materials in the current market is uneven, the market order is disordered, and even the bad condition of expelling good medicines by using inferior medicines appears. However, the existing medicinal material quality standard of the deer-horn glue is very simple, the quality control is carried out only by identifying amino acid components or only by controlling the amino acid components, the quality control lacks a special identification method and a high-sensitivity detection technology, and simultaneously, because the deer-horn glue is formed by decocting deer skin at high temperature, the protein of the deer-horn glue is denatured and damaged, and the common animal medicinal material identification technology DNA-PCR cannot identify the protein, so that the existing deer-horn glue lacks an effective true and false identification method.
In recent years, the application of mass spectrum polypeptide identification technology in identification and recognition of zoogles is more and more extensive. For example, the use of characteristic peptide fragment detection technology in the research of specific identification methods for medicinal gel materials (Chinese medicine J2015, 50 (2): 104-108) discloses the use of enzyme digestion technology and mass spectrometry identification technology to compare and research donkey-hide gelatin with new donkey-hide gelatin, oxhide gelatin and tortoise-shell gelatin, and to identify characteristic peptides, thereby establishing identification methods for several medicinal gel materials. CN105842375A discloses a group of polypeptides for identifying donkey-derived ingredients in donkey-hide gelatin and products thereof, and establishes a rapid identification method of donkey-hide gelatin and products thereof by liquid chromatography-mass spectrometry by using characteristic polypeptides.
However, the mass spectrum identification research for deer-gelatin medicinal materials and products thereof is still blank, and no complete sequence library of deer-collagen exists in the international protein sequence library, so that complete spectrum library search cannot be carried out. Therefore, there is a difficulty in establishing a method for identifying deer glue based on mass spectrometry.
Disclosure of Invention
In order to solve at least part of technical problems in the prior art, the invention analyzes the deer-horn glue and other animal glue, and discovers characteristic peptide segments capable of identifying and identifying the deer-horn glue medicinal material after systematic detection, comparison and verification, can be used for identifying the truth of the deer-horn glue, and avoids the technical defect that false positive easily occurs when only characters and appearances are used for identification at present. Specifically, the present invention includes the following.
In the first aspect of the invention, the deer-horn glue characteristic peptide is provided, when liquid chromatography-tandem mass spectrometry is adopted for multi-reaction detection in an electrospray negative ion mode, the parent ion of the deer-horn glue characteristic peptide is 732.84, and the daughter ions of the deer-horn glue characteristic peptide are 875.42 and 818.40; or the parent ion of the deer glue characteristic peptide is 675.99, and the daughter ion is 914.46, 746.38.
The deer glue characteristic peptide according to the first aspect of the invention, preferably, the conditions of the liquid chromatography-tandem mass spectrometry comprise:
taking a C18 chromatographic column as a chromatographic column, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a 0.1 volume percent formic acid aqueous solution, the mobile phase B is acetonitrile, and gradient elution is as follows: 0-10min,5-50 vol% of mobile phase B,10-15min,50 vol% of mobile phase B;
the mass spectrum was in positive ion detection mode, the atomizer temperature was 350 ℃, the atomizer flow rate was 10L/min, the atomizer pressure was 35psi, the capillary voltage was 3500V, the sheath flow gas temperature was 350 ℃, the sheath flow gas flow rate was 12L/min, the capillary voltage was 4500V, and the cone hole voltage was 500V.
In a second aspect of the invention, a deer glue characteristic peptide is provided, wherein the deer glue characteristic peptide is selected from at least one peptide with a sequence of SEQ ID No. 1 or SEQ ID No. 2.
In a third aspect of the invention, there is provided a composition for identifying whether a test sample contains deer-horn glue, comprising a deer-horn glue characteristic peptide according to the first or second aspect of the invention.
In a fourth aspect of the present invention, there is provided a method for identifying whether a sample to be tested contains deer-horn glue, which comprises the step of detecting the deer-horn glue characteristic peptide of the first or second aspect.
The method for identifying whether the deer-horn glue is contained in the sample to be tested according to the fourth aspect of the invention comprises the following steps:
(1) The treatment step of the sample to be detected comprises the following steps: taking deer gelatin product to be detected, adding ammonium solution for treatment, performing enzymolysis with trypsin, and performing enzymolysis 18 Solid phase extractionObtaining a sample solution to be detected;
(2) Performing multi-reaction detection on the presence of the deer-horn glue characteristic peptide in the sample to be detected by adopting a liquid chromatography-tandem mass spectrometry method in an electrospray negative ion mode, wherein the conditions of the liquid chromatography-tandem mass spectrometry method comprise that a C18 chromatographic column is used as a chromatographic column, a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is 0.1 volume percent formic acid aqueous solution, the mobile phase B is acetonitrile, and gradient elution is as follows: 0-10min,5-50 vol% of mobile phase B,10-15min,50 vol% of mobile phase B, mass spectrum is in a positive ion detection mode, the temperature of an atomizer is 350 ℃, the flow rate of the atomizer is 10L/min, the pressure of the atomizer is 35psi, the capillary voltage is 3500V, the temperature of a sheath flow gas is 350 ℃, the flow rate of the sheath flow gas is 12L/min, the capillary voltage is 4500V, and the conical hole voltage is 500V;
(3) If the parent ion is detected at 732.84 and the daughter ion is 875.42, 818.40; or the parent ion is 675.99, and the daughter ion is 914.46, 746.38, determining that the sample to be tested contains deer glue product.
The method for identifying whether or not deer glue is contained in a sample to be tested according to the fourth aspect of the present invention, further preferably comprises the steps of:
(1') preparation of deer-horn gelatin control solution: taking deer glue as reference substance, adding ammonium solution for treatment, performing enzymolysis with trypsin, and performing enzymolysis 18 Performing solid phase extraction to obtain deer glue control solution;
(2') preparation of a sample solution to be tested: taking a sample to be detected, and preparing a sample solution to be detected in the same manner as the step (1');
(3 ') performing multi-reaction monitoring on the deer-horn glue control solution obtained in the step (1 ') and the sample solution to be detected obtained in the step (2 ') by adopting liquid chromatography-tandem mass spectrometry and in an electrospray negative ion mode, and selecting m/z732.84 → 875.42, 818.40 or m/z675.99 → 914.46, 746.38 as detection ion pairs for detection;
(4') in the liquid chromatogram-tandem mass spectrogram of the sample to be detected, if a mass spectrum peak consistent with the retention time of the deer gum control chromatogram is present, judging that the sample to be detected contains deer gum; and if the corresponding mass spectrum peak does not appear, judging that the sample to be detected does not contain deer glue.
The method for identifying whether or not deer-horn glue is contained in a sample to be tested according to the fourth aspect of the present invention, wherein the ammonium solution is an aqueous solution of one or more selected from the group consisting of ammonium bicarbonate, ammonium acetate and ammonium formate.
According to the fourth aspect of the invention, the method for identifying whether the deer-horn glue is contained in the sample to be detected or not is provided, wherein the trypsin enzymolysis condition comprises that the enzyme concentration is 5 mug-20 mug in each 100 mug of sample liquid, the enzymolysis temperature is 30-40 ℃, and the enzymolysis time is 10-24 hours.
The method for identifying whether the deer glue is contained in the sample to be tested according to the fourth aspect of the invention, wherein the cleavage voltage for multi-reaction monitoring is 120V, and the collision energy is 35V; or the cracking voltage is 100V and the collision energy is 25V.
The deer-horn glue characteristic peptide can identify and identify deer-horn glue medicinal materials, and a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) rapid detection method taking characteristic peptide fragment ion pairs as indexes can be established on the basis of the deer-horn glue characteristic peptide. In addition, the identification method adopts the high performance liquid chromatography-tandem mass spectrometry technology, has high detection sensitivity, can be used for identifying single deer-horn glue, can also identify whether a product containing the deer-horn glue contains deer-horn glue components or not, and enlarges the application range of the technical method.
Drawings
FIG. 1 is a mass spectrometric ionization diagram of an exemplary deer-gelatin characterization peptide of the present invention.
FIG. 2 is a mass ionization diagram of another exemplary deer-gelatin characterization peptide of the present invention.
FIG. 3 is a mass spectrum of m/z732.84 → 875.42 detection of an exemplary deer-horn characteristic peptide in MRM mode.
FIG. 4 is a mass spectrum of m/z732.84 → 818.40 detection of an exemplary deer-horn glue characteristic peptide in MRM mode.
FIG. 5 is a mass spectrum of m/z675.99 → 914.46 detection of another exemplary deer glue characteristic peptide in MRM mode.
FIG. 6 is a mass spectrum of m/z675.99 → 746.38 detection of another exemplary deer glue characteristic peptide in MRM mode.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
By gelatin is meant gelatin made from animal tissue, such as from animal skin. Although animal sources vary, the main components of these animal glues are collagen. Therefore, it is difficult to effectively distinguish gelatin from animal sources at the protein level. The inventor treats animal glue from different sources with ammonium solution and then trypsin to obtain a large number of short peptide fragments, and unexpectedly discovers that partial peptide fragments exist only in the gelatin of the deer animals and not in the gelatin of other mammals or zymolytes thereof when the peptide fragment identification is carried out by a high performance liquid chromatography-tandem mass spectrometry rapid detection method (HPLC-MS/MS). The present invention has been accomplished, at least in part, based on this.
The "deer glue" according to the present invention refers to gelatin prepared from the skin of a cervidae animal, and thus, is sometimes referred to as "deer skin glue". Methods for preparing deer gelatin are known in the art. In addition, the donkey-hide gelatin can be prepared by referring to the process for preparing donkey-hide gelatin. Examples of Cervidae animals include, but are not limited to, cervus elaphus Linnaeus or Cervus nippon Temminck.
The "deer glue characteristic peptide" includes the first deer glue characteristic peptide and the second deer glue characteristic peptide which are described later, and means a peptide which is only present in deer glue but not present in other animals, particularly in glue made from the skin of other animals. In preferred embodiments, examples of other animals include donkeys, cattle, pigs, horses, and sheep. Therefore, the deer-horn characteristic peptide of the present invention is preferably not present in at least donkey-hide gelatin (donkey-hide gelatin), bovine hide gelatin (yellow gelatin), porcine hide gelatin (neogelatin), equine hide gelatin and ovine hide gelatin, nor in decomposition products of these gums. I.e., the deer glue characteristic peptide is not a fragment of the proteins in these glues. Preferably, the protease (e.g., trypsin) does not enzymatically cleave the deer-gelatin characteristic peptide of the invention. Preferably, the deer glue characteristic peptide of the invention is a small molecule peptide fragment, such as a peptide fragment consisting of 10-35 amino acids.
[ first deer-horn glue characteristic peptide ]
In a first aspect of the invention, there is provided a first deer-horn glue characteristic peptide, which is a characteristic peptide characterized by liquid chromatography-tandem mass spectrometry. Specifically, when multiple reaction detection was performed by liquid chromatography-tandem mass spectrometry in electrospray negative ion mode, it was only shown to be present in deer-horn gelatin, at least not in donkey-hide gelatin (ass-hide gelatin), bovine hide gelatin (oxhide gelatin), porcine hide gelatin (neo-ass-hide gelatin), equine hide gelatin, and ovine hide gelatin. The first deer characteristic peptide can be a peptide segment or a combination of a plurality of peptide segments. Preferably, the first deer-horn glue characteristic peptide of the invention is a composition of a plurality of peptide fragments. E.g., a combination of at least two different peptide fragments. In an exemplary embodiment, the first deer-horn glue characteristic peptide includes peptide fragment 1 and peptide fragment 2. For peptide fragment 1, when the liquid chromatography-tandem mass spectrometry is adopted to carry out multi-reaction detection in an electrospray negative ion mode, the maximum response value is obtained at m/z732.84 within the m/z100-2000 full-scan range of the primary mass spectrum. That is, the m/z value of the parent ion of peptide fragment 1 was 732.84. Within the m/z100-2000 full scan range of the secondary mass spectrum, there are the two largest response values at m/z875.42, m/z 818.40. Namely, the m/z values of the daughter ions of peptide fragment 1 were 875.42 and 818.40. In addition, for peptide fragment 2, the maximum response value is obtained at m/z675.99 within the full scan range of m/z100-2000 of the primary mass spectrum. Namely, the m/z value of the parent ion of peptide fragment 2 was 675.99. The two response values are greatest at m/z914.46, 746.38 over the full scan range of m/z100-2000 of the secondary mass spectrum. That is, the m/z values of the daughter ions of peptide fragment 2 were 914.46 and 746.38.
In certain embodiments, the conditions of the liquid chromatography-tandem mass spectrometry of the present invention comprise: with C 18 The chromatographic column is a chromatographic column, the mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is 0.1 volume percent formic acid aqueous solution, the mobile phase B is acetonitrile, and gradient elution is as follows: 0-10min,5-50 vol% of mobile phase B,10-15min and 50 vol% of mobile phase B. The mass spectrum was in positive ion detection mode, the atomizer temperature was 350 ℃, the atomizer flow rate was 10L/min, the atomizer pressure was 35psi, the capillary voltage was 3500V, the sheath flow gas temperature was 350 ℃, the sheath flow gas flow rate was 12L/min, the capillary voltage was 4500V, and the cone voltage was 500V.
[ second deer glue characteristic peptide ]
In a second aspect of the invention, there is provided a second deer-horn gelatin signature peptide, which is a signature peptide characterized by an amino sequence. Preferably, the second deer glue characteristic peptide comprises peptide fragment A and peptide fragment B. Wherein the amino acid sequence of the peptide segment A is shown as SEQ ID No. 1, and the amino acid sequence of the peptide segment B is shown as SEQ ID No. 2. Like the first deer-horn glue characteristic peptide, the second deer-horn glue characteristic peptide can be peptide segment A alone or peptide segment B alone, or a combination of peptide segment A and peptide segment B.
In certain embodiments, the second deer-horn glue characteristic peptide of the invention also has the characteristics described for the first deer-horn glue characteristic peptide. For example, the amino acid sequence of peptide fragment A is shown in SEQ ID No. 1, and the m/z value of the parent ion is 732.84, and the m/z value of the daughter ion is 875.42, 818.40. As another example, the amino acid sequence of peptide fragment B is shown in SEQ ID No. 2, and the parent ion m/z value is 675.99, and the daughter ion m/z values are 914.46, 746.38.
[ composition for identifying whether deer-horn glue is contained in a sample to be tested ]
In a third aspect of the invention, there is provided a composition for identifying deer-horn glue (sometimes referred to simply as "the composition of the invention") comprising a first deer-horn glue characteristic peptide or a second deer-horn glue characteristic peptide of the invention as a reference or internal standard. The reference or internal standard of the present invention refers to a substance which is a comparative object in the identification of a deer-horn gelatin product. The composition can be used for judging whether a sample to be detected is a deer-horn glue product or not, and can also be used for detecting whether the sample to be detected contains deer-horn glue components or the content of the deer-horn glue components.
The composition of the invention may also contain other ingredients and/or means for detecting deer-horn glue. Examples of other components include, but are not limited to, ammonium solutions, trypsin, reagents used for mass spectrometric detection (e.g., mobile phase), and the like. Examples of devices include, but are not limited to, chromatography columns such as C 18 A chromatography column, vial or vial. Other ingredients or devices may employ products or reagents known in the art.
[ method for identifying whether deer-horn glue is contained in a sample to be tested ]
In a fourth aspect of the invention, there is provided a method (sometimes referred to as the method of the invention) for identifying the presence of deer-horn glue in a sample to be tested, comprising the step of detecting a first or second deer-horn glue characteristic peptide of the invention. The method for identifying whether the sample to be detected contains the deer-horn glue comprises the steps of identifying whether the sample to be detected is the deer-horn glue, namely identifying the truth of the deer-horn glue, and identifying whether the sample to be detected contains the deer-horn glue.
In an exemplary authentication method, the method of the invention comprises the steps of:
(1) And (3) processing a sample to be detected: adding ammonium solution into sample to be detected, treating, performing enzymolysis with trypsin, and performing enzymolysis 18 Performing solid phase extraction to obtain a sample solution to be detected;
(2) Performing multi-reaction detection on the presence of the deer-horn characteristic peptide in the sample to be detected by adopting a liquid chromatography-tandem mass spectrometry method in an electrospray negative ion mode, wherein the conditions of the liquid chromatography-tandem mass spectrometry method comprise C 18 The chromatographic column is a chromatographic column, the mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is 0.1 volume percent formic acid aqueous solution, the mobile phase B is acetonitrile, and the gradient elution is as follows: 0-10min,5-50 vol% of mobile phase B,10-15min,50 vol% of mobile phase B, a mass spectrum in a positive ion detection mode, an atomizer temperature of 350 ℃, an atomizer flow rate of 10L/min, an atomizer pressure of 35psi, a capillary voltage of 3500V, a sheath flow gas temperature of 350 ℃, a sheath flow gas flow rate of 12L/min, a capillary voltage of 4500V, and a cone hole voltage of 500V;
(3) If the parent ion is detected at 732.84 and the daughter ion is 875.42, 818.40; or the parent ion is 675.99, and the daughter ion is 914.46, 746.38, determining that the sample to be tested contains deer glue.
In a further exemplary identification method, the method of the invention comprises the steps of:
(1') preparation of deer-horn gelatin control solution: taking deer glue as reference substance, adding ammonium solution for treatment, performing enzymolysis with trypsin, and performing enzymolysis 18 Performing solid phase extraction to obtain deer glue control solution;
(2') preparation of a sample solution to be tested: taking a sample to be detected, and preparing a sample solution to be detected in the same manner as the step (1);
(3 ') detecting the deer glue control solution obtained in the step (1 ') and the sample solution to be detected obtained in the step (2 ') by adopting liquid chromatography-tandem mass spectrometry and adopting an electrospray negative ion mode to carry out multi-reaction monitoring, and selecting m/z732.84 → 875.42, 818.40 and/or m/z675.99 → 914.46 and 746.38 as detection ion pairs;
(4') in the liquid chromatogram-tandem mass spectrogram of the sample to be detected, if a mass spectrum peak consistent with the retention time of the deer gum control chromatogram is present, judging that the sample to be detected contains deer gum; and if the corresponding mass spectrum peak does not appear, judging that the sample to be detected does not contain deer-horn glue.
The reagents or conditions used in the two exemplary methods described above are the same and are applicable to all other similar methods of identifying whether deer-horn gelatin is included in the sample to be tested.
The ammonium solution of the present invention is a solution for treating deer-horn glue, and preferably an aqueous solution of inorganic ammonium. The invention discovers that after the animal glue is treated by ammonium solution, the animal glue is subjected to trypsin enzymolysis to obtain peptide fragments capable of distinguishing the deer glue from other animal glue. Wherein the pH of the ammonium solution is generally 7 to 8, preferably 7.4 to 7.8. Preferably, the ammonium solution is an aqueous solution of one or more substances selected from the group consisting of ammonium bicarbonate, ammonium acetate and ammonium formate. The concentration of inorganic ammonium in the ammonium solution is generally 1 to 10% by weight, preferably 2 to 8% by weight, more preferably 5 to 6% by weight. The higher the concentration of the ammonium solution, the more favorable the enzymolysis is to obtain the required characteristic peptide. The amount of glue used is 0.1-5g, preferably 0.2-3g, per 100ml of ammonium solution. The ammonium solution treatment time is generally 5 minutes to 5 hours, preferably 10 minutes to 60 minutes. In certain embodiments the ammonium solution is treated in a sealed environment at a temperature of 30 to 50 c, preferably 35 to 45 c.
The trypsin of the present invention may be a known enzyme. The conditions of the enzymatic hydrolysis of the present invention are important. It comprises that each 100 μ l sample liquid contains 5 μ g-20 μ g, preferably 10-15 μ g trypsin, the enzymolysis temperature is 30-40 deg.C, preferably 35-38 deg.C, more preferably 37 deg.C, and the enzymolysis time is 10-24 hours, preferably 15-20 hours.
The cracking voltage of the multi-reaction monitoring is 120V, and the collision energy is 35V; or the cracking voltage is 100V and the collision energy is 25V.
Example 1
This example is the screening and identification of deer glue characteristic peptide, the specific method is as follows:
1. reagent and apparatus
Mass spectrometry reagents such as Sodium Dodecyl Sulfate (SDS), dithiothreitol (DTT), ammonium bicarbonate, trypsin (Promega Mass Spectrometry), acetonitrile, methanol, and formic acid were mass spectrometry (Merck, germany).
Nano-scale liquid chromatography system: eksigentekspert TM NANOLC LIQUID PHASE SYSTEM, eksiginsoftware V.3.12 LIQUID PHASE WORKING STATION. High resolution mass spectrometry system: tripleTOF5600, equipped with electrospray ionization source (ESI), analyst TF1.6 chromatographic workstation, peak view1.2 mass spectrometry software and protein analysis software proteinpilot4.5 (AB Sciex usa). Centrifugal concentrator (Labconco, USA); zipTipC18 Pipette Tips (Millipore, USA).
2. Glue medicinal material sample
Collecting colla Cornus Cervi (deer skin gelatin), colla Corii Asini, corii bovis Seu Bubali (oxhide gelatin), corii Sus Domestica (new colla Corii Asini), corii equi Domestica, corii Caprae Seu Ovis, etc., and collecting specific information in the following table.
TABLE 1 gum drug information
3. Enzymolysis of glue medicinal materials
Taking 0.1g of various gum medicinal materials, placing in 50ml conical flask, adding 1% NH 4 HCO 3 Fixing volume of the solution to scale, dissolving, preparing trypsin solution (dissolving pancreatin 100 μ g with Resuspension Buffer1 ml), adding gel liquid medicine 100 μ l into prepared trypsin solution 10 μ l, performing enzymolysis at 37 deg.C for 12h, incubating at 90 deg.C for 5min for inactivation, centrifuging (10000 r/min), and inactivating with C 18 Solid phase extraction desalination, drying sample liquid with nitrogen, adding water for dissolution, and reserving.
4. Chromatography analysis
Eksigentekspert TM nano-LC liquid phase system, the chromatographic column is 5 mu m Reprosil C 18 AQ (75 μm. Times.150 mm), LC-MS/MS system analyzed different gel samples. The loading was 5 μ L, flow rate 400nL/min, mobile phase a (acetonitrile/formic acid/water =2/0.2/98, v/v/v),mobile phase B (acetonitrile/formic acid/water =80/0.2/20, v/v/v), 2-30% B linear gradient elution for 60min.
AB SCIEXTripleTOF5600, ESI source, positive ion scan, spray voltage 2.5kV, ion transport capillary temperature 200 ℃; the primary full-scan range of the mass spectrum is m/z100-2000, and the separation width is 3Da; the tandem mass spectrometry adopts a secondary mass spectrometry scanning mode depending on primary mass spectrometry data, and 5 ions with the highest ion intensity in the primary mass spectrometry are sequentially selected for carrying out Collision Induced Dissociation (CID) secondary tandem mass spectrometry. Data analysis was performed using the ProteinPilot protein analysis software. Selecting Lauraiateria database (Uniprot database download), setting the search parameters as follows: error of 10ppm for precursor ion; a daughter ion error of 1Da; allowing 2 sites to be cut by mistake, wherein the false positive rate (FDR) is less than or equal to 1 percent; selecting pancreatin (Trypsin) in a digestion mode, wherein the number of unique peptide segments (unique peptides) is more than or equal to 2; the other parameters are default parameters, and the scores obtained under the searching conditions have significance (P < 0.05) and are considered as effective identification results.
Polypeptides were identified from the samples of table 1: wherein 280-611 peptide fragments are identified in the deer-horn glue sample; 183-326 peptide fragments are identified in the donkey-hide gelatin; 365-513 peptide fragments are identified in the oxhide glue; 348-510 peptide fragments are identified in the pigskin glue; 181-502 peptide fragments are identified in the equine hide glue; 169 to 466 peptide fragments are identified in the sheep skin glue;
the strategy for screening and determining the characteristic peptide fragments comprises the following 3 steps: (1) comparing and determining the information of the common peptide segments in the deer skin glue sample as a set A; (2) respectively taking all the peptide fragment information in different types of sample gelatin (such as taking and collecting peptide fragments of different batches of donkey-hide gelatin samples) as a set B; (3) and (3) performing cross analysis on the set A and the set B, wherein the components excluding the intersection of the A and the B in the set A are specific components of the set A, and analyzing each specific component to obtain the specific polypeptide in the deer-horn glue.
5. Identification and structural analysis of characteristic peptide fragment sequence
And (3) performing library searching identification analysis on the secondary mass spectrum data by adopting PEAKS 7.5 software, wherein the searching parameters are set as follows: error of 10ppm for precursor ion; a daughter ion error of 1Da; cysteine residue fixation modification (carbamoylation 57.02 Da); variable modification of methionine residues (oxidation +15.99 Da); n-terminal acetylation (+ 42.01 Da); carbamoylation (+ 43.01 Da); allowing 2 sites to be cut by mistake, wherein the false positive rate (FDR) is less than or equal to 1 percent; selecting Trypsin enzyme (Trypsin); the other parameters are default parameters, and the scores obtained under the searching conditions have significance (P < 0.05) and are considered as effective identification results.
The characteristic peptide fragment is identified in the figures of fig. 1 and fig. 2, and the sequence of the peptide fragment is shown in table 2. The peptide with the sequence SEQ ID No. 1 is also called peptide fragment I, and the peptide with the sequence SEQ ID No. 2 is also called peptide fragment II.
TABLE 2
| Serial number | Peptide fragment sequence | Molecular weight | m/z | Source |
| SEQ ID No:1 | SGETGASGPP(Hyp)GFAGEK | 1463.65 | 732.84 | Deer glue |
| SEQ ID No:2 | GEVGPAGPDGFAGPAGAAGQSGAK | 2024.96 | 675.99 | Deer glue |
Example 2
This example is the identification of deer glue and other glue medicinal materials based on characteristic peptide
1. Reagent and apparatus
Sodium Dodecyl Sulfate (SDS), dithiothreitol (DTT), ammonium bicarbonate, trypsin (Promega Mass Spectrometry grade), acetonitrile, methanol, formic acid and other mass spectrometry reagents are mass spectrometry grade (Merck, germany).
High resolution fast liquid chromatography tandem triple quadrupole mass spectrometry (AB corporation, usa). Centrifugal concentrator (Labconco, USA); ziptipC 18 PipetateTips (Millipore, USA).
2. Deer glue and other glue medicinal materials
Collecting colla Cornus Cervi (deer skin gelatin), colla Corii Asini, corii bovis Seu Bubali (oxhide gelatin), corii Sus Domestica (new colla Corii Asini), corii equi Domestica, and Corii Caprae Seu Ovis, etc., as shown in Table 1.
The deer glue reference medicine is prepared from sika deer skin.
3. Sample processing
Taking 0.lg of each glue medicinal material powder, adding 50ml of ammonium acetate solution (3%) with pH =7.5, heating and ultrasonically treating for 30 minutes, filtering by using a microporous membrane, taking 100 mu L of liquid medicine, placing the liquid medicine into a microscale sample injection bottle, adding 10 mu L of trypsin solution (taking trypsin for sequence analysis, adding 1% ammonium bicarbonate solution to prepare a solution containing lmg in each lml, preparing the solution temporarily), shaking uniformly, carrying out enzymolysis at the constant temperature of 37 ℃ for 12 hours, and carrying out enzymolysis by using C 18 Solid phase extraction desalination, drying sample liquid with nitrogen, adding water for dissolution, and reserving. Negative samples were prepared in the same manner.
3. Detection conditions
Liquid phase conditions: the chromatographic column is Waters C 18 (2.1 mm. Times.50mm, 1.8 μm), a flow rate of 0.3ml/min, a mobile phase of 0.1% aqueous formic acid as mobile phase A, and acetonitrile as mobile phase B, and gradient elution was carried out: 0-10min, 5-50% by weight B;10-15min,50% B;
mass spectrum conditions: ESI positive ion detection mode, nebulizer temperature: 350 ℃, atomizer flow rate: 10L/min, atomizer pressure 35psi, capillary voltage: 3500V, sheath flow gas temperature: 350 ℃, sheath flow rate: 12L/min, capillary voltage 4500V, taper hole voltage: 500V.
TABLE 3 Multiple Reaction Monitoring (MRM) mode conditions
4. Specificity experiments
Respectively taking a deer skin gelatin reference medicinal material as a reference and deer skin gelatin, donkey-hide gelatin, oxhide gelatin, pigskin gelatin, horse hide gelatin and sheep hide gelatin as samples, and selecting deer skin gelatin characteristic ion pairs for determination.
TABLE 4 detection results of deer glue and other various glue medicinal materials
Note: "+" indicates detection of a chromatographic peak; "-" indicates no significant chromatographic peak was detected.
The result shows that in the liquid chromatogram-mass spectrogram of the sample to be detected, two chromatographic peaks consistent with the chromatographic retention time of the deer-gelatin control medicinal material sample are simultaneously presented in five batches of deer-gelatin samples, but no corresponding chromatographic peak is detected in other various counterfeit gelatin samples. The deer-horn glue characteristic peptide and the detection method can well identify the deer-horn glue and other glue medicinal materials.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
SEQUENCE LISTING
<110> Guangji Tang pharmaceutical Co., ltd, guizhou
<120> deer glue characteristic peptide and method for identifying deer glue contained in sample to be detected
<130> 180080011ZC
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> PRT
<213> Cervus nippon Temminck
<400> 1
Ser Gly Glu Thr Gly Ala Ser Gly Pro Hyp Gly Phe Ala Gly Glu Lys
1 5 10 15
<210> 2
<211> 24
<212> PRT
<213> Cervus nippon Temminck
<400> 2
Gly Glu Val Gly Pro Ala Gly Pro Asp Gly Phe Ala Gly Pro Ala Gly
1 5 10 15
Ala Ala Gly Gln Ser Gly Ala Lys
20
Claims (2)
1. A deer glue characteristic peptide is characterized in that the deer glue characteristic peptide is a peptide with SEQ ID No. 2.
2. A composition for identifying whether or not a deer-horn glue is contained in a sample to be tested, comprising the deer-horn glue characteristic peptide according to claim 1.
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| CN112098579B (en) * | 2020-09-01 | 2021-11-23 | 南京中医药大学 | Characteristic peptide segment for distinguishing deerhorn glue or deerhorn glue and detection method thereof |
| CN113173971B (en) * | 2021-04-20 | 2022-08-23 | 江阴天江药业有限公司 | Characteristic peptide segment for identifying formula granules containing deer horns or deer skins and detection method thereof |
| CN113804897A (en) * | 2021-09-17 | 2021-12-17 | 中国食品药品检定研究院 | Sequence identification method for characteristic peptide segments of cordyceps sinensis and various fermented cordyceps sinensis preparations |
| CN114855285B (en) * | 2022-06-02 | 2023-02-17 | 山东省食品药品检验研究院 | Characteristic polypeptide library for rapidly identifying species source of pilose antler and application thereof |
| CN115792243A (en) * | 2022-11-25 | 2023-03-14 | 北京同仁堂(辽宁)科技药业有限公司 | Method for detecting deer glue by using special ion pairs and application |
| CN117106031B (en) * | 2023-10-20 | 2024-01-02 | 山东省食品药品检验研究院 | Common characteristic peptide segment of reindeer horn, camel horn and deer horn and application thereof |
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| CN103424546B (en) * | 2012-05-18 | 2015-03-11 | 苏州红冠庄国药股份有限公司 | Specific identification method for biological active components in deer leather gel |
| CN103630620B (en) * | 2013-10-28 | 2015-06-24 | 山东东阿阿胶股份有限公司 | Method for detecting deer-derived ingredients in glue type traditional Chinese medicines and products thereof |
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