Screening method of characteristic peptides among different species and application of screening method

Abstract

The invention discloses a method for screening characteristic peptides among different species. The screening method effectively solves the problem of misjudgment or missed judgment of species characteristic peptides caused by lack of species protein database information and similar species among species. The characteristic peptide combination for identifying the tortoise-shell glue component is obtained on the basis of the screening method and comprises the characteristic peptide with the amino acid sequence as shown in SEQ ID NO: 1-45; the characteristic peptide combination can be used for effectively identifying the existence of a grass turtle source substance, a Brazilian turtle source substance, a flower turtle source substance, a pig source substance and / or a bovine source substance in the tortoise-shell glue, so that source species of the tortoise-shell glue can be traced.

Description

Technical Field

[0001] The present invention relates to the field of biological testing technology, in particular to a method for screening characteristic peptides between different species and an application thereof. Background Art

[0002] Tortoise shell collagen is an important raw material in tortoise glue (tortoise shell glue, tortoise plastron glue or tortoise longevity glue) and tortoise jelly, two popular health foods in China. Its authenticity and quality issues have attracted widespread public attention. Tortoise glue and tortoise jelly have the effects of nourishing yin, replenishing blood and stopping bleeding. Modern Chinese medicine believes that such foods have the effects of replenishing yin deficiency, enhancing immunity and prolonging life.

[0003] Turtle glue is mainly made from tortoise shell. The main active ingredient is the active peptide hydrolyzed from the collagen in the tortoise shell. The tortoise shell used can only be boiled with Chinese grass tortoise shell and a small amount of flower tortoise shell. Some illegal merchants use some cheap collagen to make tortoise glue in order to make profits. Common adulterants include pig skin, cowhide, turtle meat and Brazilian tortoise shell, which has also led to frequent adulteration and inferior products in the current tortoise glue market.

[0004] Therefore, tracing the species origin of food substances is particularly important. Currently, commonly used methods for tracing the species origin of food substances include DNA barcoding, polymerase chain reaction (PCR), and high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Although DNA barcoding and PCR can detect species information in samples, DNA degradation in highly processed foods can lead to cross-contamination and low DNA extraction during pre-extraction. Compared to DNA technology, peptides are more stable during processing, especially in foods processed at high temperatures. Even with partial protein degradation, characteristic peptides of a species can still be detected. UPLC-MS / MS technology uses ultra-high performance liquid chromatography as a separation system and mass spectrometry as a detection system. The sample is separated from the mobile phase and then subjected to mass spectrometry. The mass spectrometer separates the ion fragments according to their mass number, and the detector generates mass spectral data. Based on the mass spectral data and the theoretical protein sequence in the species protein database, precise matching is performed to obtain information about the peptides in the sample and their source protein, thereby determining the characteristic peptides of the sample's source species.

[0005] There are two main challenges in the current research on characteristic peptides. First, the current research on characteristic peptides relies on the integrity and accuracy of the online species protein database. During the identification process of peptides, it is necessary to refer to the protein sequence information in the database to ensure that the identified peptides are indeed specific to the target species. If the protein database of a certain species is incomplete, it will lead to inaccuracy and incompleteness in peptide detection, thereby affecting the identification of characteristic peptides and further affecting the traceability of the source species of food substances. Secondly, in the process of characteristic peptide research, it is necessary to use bioinformatics tools such as BLAST to manually identify characteristic peptides. Currently, it is usually based on finding peptides that fully match the target species and are not matched in other species. However, just because a peptide exists in the target species and has not been found in other known substances, it cannot completely rule out the possibility that the peptide exists in species not recorded in the database. This may lead to oversimplification and misjudgment of characteristic peptides, thereby affecting the traceability of the source species of food substances.

[0006] Therefore, there is a need for an identification and screening method that can target species characteristic peptides, obtain relative characteristic peptides between different species, and thus ensure the accuracy of species traceability of food material sources. Summary of the Invention

[0007] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a method for screening characteristic peptides between different species and its application.

[0008] The first object of the present invention is to provide a method for screening characteristic peptides among different species.

[0009] The second object of the present invention is to provide the application of the above screening method in screening characteristic peptides among different species.

[0010] The third object of the present invention is to provide a characteristic peptide combination for identifying the components of tortoise shell glue.

[0011] The fourth object of the present invention is to provide the use of the above-mentioned characteristic peptide combination in identifying the components of tortoise shell glue.

[0012] The fifth object of the present invention is to provide the use of the above-mentioned characteristic peptide combination as a quantitative marker in the quantitative detection of tortoise shell glue components.

[0013] The sixth object of the present invention is to provide a method for identifying the components of tortoise shell glue.

[0014] In order to achieve the above object, the present invention is implemented through the following scheme:

[0015] A method for screening characteristic peptides between different species, using at least two different species as the species to be detected, comprises the following steps:

[0016] S1. Obtain protein sequence information of the species to be detected from the protein database;

[0017] When the number of protein sequence information of the species to be detected in the protein database is less than 1000, the protein sequence information of the species to be detected in the protein database after removing the species other than the species to be detected from the genus to which the species to be detected belongs is used as the protein sequence information of the species to be detected; when the number of protein sequence information of the species to be detected in the protein database is less than 1000, the protein sequence information of the species to be detected in the protein database is used as the protein sequence information of the species to be detected;

[0018] S2. Performing theoretical enzyme digestion on the protein sequence information of the species to be detected obtained in step S1 to obtain theoretical enzyme-digested peptides of the species to be detected, and recording the source protein of the theoretical enzyme-digested peptides of the species to be detected;

[0019] S3. Screening the theoretical enzyme-cleaved peptides of the species to be detected obtained in step S2 to obtain differential peptides of the species to be detected;

[0020] The screening specifically includes: removing peptides with a length of less than 4 aa or greater than 20 aa in the theoretical enzyme-cleaved peptides of the species to be detected, removing peptides that appear repeatedly in the theoretical enzyme-cleaved peptides of different species to be detected, and deduplicating peptides that appear repeatedly in the theoretical enzyme-cleaved peptides of the species to be detected;

[0021] S4. Selecting peptides that meet the selection criteria from the differential peptides of the species to be detected obtained in step S3 as characteristic peptides of the species to be detected;

[0022] The selection conditions are: the differential peptide segment of the species to be detected does not exist in the protein sequence information of the species to be detected other than the species to be detected, the source protein of the differential peptide segment of the species to be detected exists in the protein of the species to be detected other than the species to be detected, and the sequence identity between the differential peptide segment of the species to be detected and the peptide segment in the species to be detected other than the species to be detected is not less than 50%;

[0023] The peptides in the species to be detected other than the species to be detected are derived from the same protein as the differential peptides in the species to be detected, and their positions on the protein are offset by no more than 20 aa from the positions of the differential peptides in the species to be detected on the protein.

[0024] Preferably, the protein database in step S1 is the NCBI database, the Uniprot database and / or the PDB database.

[0025] More preferably, the protein database in step S1 is the NCBI database.

[0026] Preferably, the protein sequence information of the species to be detected is protein sequence information in FASTA file format.

[0027] Preferably, the theoretical enzymatic digestion in step S2 is theoretical enzymatic digestion by trypsin and / or theoretical enzymatic digestion by pepsin.

[0028] More preferably, the theoretical enzymatic digestion in step S2 is theoretical enzymatic digestion by trypsin.

[0029] Preferably, the selection conditions in step S4 are: the differential peptide segment of the species to be detected does not exist in the protein sequence information of the species to be detected other than the species to be detected, the source protein of the differential peptide segment of the species to be detected exists in the protein of the species to be detected other than the species to be detected, and the sequence identity between the differential peptide segment of the species to be detected and the peptide segment in the species to be detected other than the species to be detected is not less than 70%.

[0030] The present invention also seeks to protect the use of any of the above-mentioned screening methods in screening characteristic peptides among different species.

[0031] The present invention also claims protection for a characteristic peptide combination for identifying tortoise shell glue components, including characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 1 to 45.

[0032] Preferably, the characteristic peptides as shown in SEQ ID NOs: 1 to 14 are characteristic peptides of grass turtles; the characteristic peptides as shown in SEQ ID NOs: 15 to 20 are characteristic peptides of red-eared sliders; the characteristic peptides as shown in SEQ ID NOs: 21 to 25 are characteristic peptides of spotted turtles; the characteristic peptides as shown in SEQ ID NOs: 26 are characteristic peptides of pigs; the characteristic peptides as shown in SEQ ID NOs: 27 to 31 are characteristic peptides of cattle; the characteristic peptides as shown in SEQ ID NOs: 32 to 35 are common characteristic peptides of grass turtles and spotted turtles; the characteristic peptide as shown in SEQ ID NO: 36 is a common characteristic peptide of red-eared sliders, spotted turtles, pigs and cattle; the characteristic peptides as shown in SEQ ID NOs: 37 to 45 are common characteristic peptides of pigs and cattle.

[0033] The present invention also claims protection for the use of the above-mentioned characteristic peptide combination in identifying the components of tortoise shell glue.

[0034] Preferably, the identification of tortoise shell glue components is to identify the presence or absence of tortoise-derived substances, red-eared slider-derived substances, spotted turtle-derived substances, pig-derived substances and / or bovine-derived substances in the tortoise shell glue.

[0035] The present invention also seeks to protect the use of the above-mentioned characteristic peptide combination as a quantitative marker in the quantitative detection of tortoise shell glue components.

[0036] Preferably, the quantitative detection of tortoise shell glue components is the quantitative detection of tortoise-derived substances, red-eared slider-derived substances, spotted turtle-derived substances, pig-derived substances and / or bovine-derived substances in tortoise shell glue.

[0037] The present invention also claims a method for identifying the components of tortoise shell glue, comprising the following steps:

[0038] S11. Using the tortoise shell glue to be tested as a sample, ultra-performance liquid chromatography-high-resolution mass spectrometry is used to obtain mass spectrometry results, and based on the mass spectrometry results, peptide information of the tortoise shell glue to be tested is obtained;

[0039] S12. The peptide information of the tortoise shell glue to be detected obtained in step S11 is combined with the above-mentioned characteristic peptide combination for identification to obtain the identification result of the tortoise shell glue to be detected;

[0040] The identification is specifically performed by combining the above-mentioned characteristic peptide combination: the peptide information of the tortoise shell glue to be detected obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 1 to 14, and the tortoise shell glue to be detected contains tortoise-derived substances;

[0041] The peptide information of the tortoise shell glue to be tested obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 15 to 20, indicating that the tortoise shell glue to be tested contains a substance derived from red-eared turtles;

[0042] The peptide information of the tortoise shell glue to be tested obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 21 to 25, indicating that the tortoise shell glue to be tested contains tortoise-derived substances;

[0043] The peptide information of the tortoise shell glue to be tested obtained in step S11 contains a characteristic peptide with an amino acid sequence as shown in SEQ ID NO: 26, indicating that the tortoise shell glue to be tested contains porcine-derived substances;

[0044] The peptide information of the tortoise shell glue to be tested obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 27 to 31, indicating that the tortoise shell glue to be tested contains bovine-derived substances;

[0045] The peptide information of the tortoise shell glue to be tested obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 32 to 35, and the tortoise shell glue to be tested contains tortoise-derived substances and / or tortoise-derived substances;

[0046] The peptide information of the tortoise shell glue to be tested obtained in step S11 contains a characteristic peptide with an amino acid sequence as shown in SEQ ID NO: 36, and the tortoise shell glue to be tested contains a red-eared turtle-derived substance, a turtle-derived substance, a porcine-derived substance, and / or a bovine-derived substance;

[0047] The peptide information of the tortoise shell glue to be detected obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 37 to 45, and the tortoise shell glue to be detected contains porcine-derived substances and / or bovine-derived substances.

[0048] Preferably, the chromatographic conditions of the ultra-high performance liquid chromatography-high-resolution mass spectrometry technique in step S11 are: a flow rate of 0.3 μL / min, an injection volume of 2 μL, mobile phase A is a 0.1% formic acid aqueous solution with a volume concentration, and mobile phase B is a 0.1% formic acid aqueous solution containing 80% acetonitrile with a volume concentration;

[0049] The mass spectrometry conditions were as follows: spray voltage in positive ion mode, ion transfer tube temperature of 250°C, sheath gas flow rate of 5 arb, auxiliary gas flow rate of 2 arb, S-lens voltage of 50 V, heating temperature of 250°C, and scanning range of 150-2000 m / z.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The present invention provides a method for screening characteristic peptides between different species. This method effectively addresses the problem of misidentification or omission of species characteristic peptides due to a lack of information in species protein databases and the similarity of species. Furthermore, based on this screening method, the present invention has developed a characteristic peptide combination for identifying components of tortoise shell glue, including characteristic peptides with amino acid sequences represented by SEQ ID NOs: 1-45. This characteristic peptide combination can effectively identify the presence of tortoise shell glue derived materials, including materials derived from red-eared sliders, red-eared sliders, spotted turtles, porcine materials, and / or bovine materials, thereby tracing the source species of the tortoise shell glue. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Flow chart of the screening method shown in Example 1. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0054] Example 1 A method for screening characteristic peptides between different species

[0055] No less than 2 different species are used as the species to be detected, and the characteristic peptides between the species to be detected are screened. The flow chart of the screening method is as follows: Figure 1 As shown, the details are as follows:

[0056] S1. Obtain protein sequence information of the species to be detected in FASTA file format from the NCBI database, Uniprot database, and PDB database;

[0057] When the protein sequence information of the species to be detected in the NCBI database, Uniprot database and PDB database is less than 1000, the protein sequence information of the genus of the species to be detected after removing the species other than the species to be detected is used as the protein sequence information of the species to be detected; when the protein sequence information of the species to be detected in the protein database is less than 1000, the protein sequence information of the species to be detected in the protein database is used as the protein sequence information of the species to be detected;

[0058] S2. Performing theoretical trypsin digestion on the protein sequence information of the species to be detected obtained in step S1 to obtain theoretical enzyme-digested peptides of the species to be detected, and recording the source protein of the theoretical enzyme-digested peptides of the species to be detected;

[0059] S3. Screening the theoretical enzyme-cleaved peptides of the species to be detected obtained in step S2 to obtain differential peptides of the species to be detected;

[0060] The screening is specifically performed as follows: removing peptides with a length of less than 4 aa or a length of more than 20 aa from the theoretical enzyme-cleaved peptides of the species to be detected, removing peptides that appear repeatedly in the theoretical enzyme-cleaved peptides of different species to be detected, and deduplicating peptides that appear repeatedly in the theoretical enzyme-cleaved peptides of the species to be detected;

[0061] S4. Selecting peptides that meet the selection criteria from the differential peptides of the species to be detected obtained in step S3 as characteristic peptides of the species to be detected;

[0062] The selection conditions are as follows: the differential peptide of the species to be detected does not exist in the protein sequence information of the species to be detected other than the species to be detected; the source protein of the differential peptide of the species to be detected exists in the protein of the species to be detected other than the species to be detected; and the sequence identity between the differential peptide of the species to be detected and the peptide in the species to be detected other than the species to be detected is not less than 70%;

[0063] The peptides in the species to be detected other than the species to be detected are: the differential peptides in the species to be detected are derived from the same protein, and the position on the protein is offset by no more than 20 aa from the position of the differential peptide in the species to be detected on the protein.

[0064] Example 2 Screening of a Characteristic Peptide Combination for Identifying Tortoise Shell Glue Components

[0065] Tortoise shell glue is often obtained by boiling tortoise shell and tortoise shell. Common adulterants include pig skin, cow skin, and red-eared turtle shell. Therefore, this example uses tortoise shell, red-eared turtle, pig, cattle, and red-eared turtle as the species to be tested. The characteristic peptides between the above five species are detected to identify the components of tortoise shell glue. The specific details are as follows:

[0066] 1. Experimental methods

[0067] According to the method shown in step S1 of Example 1, protein sequence information (FASTA file format) of Chinese tortoise, Chinese flower turtle, pig, cattle and red-eared slider were obtained from proteins respectively;

[0068] Among them, the protein sequence information of the grass turtle contains 67,929 proteins; the protein sequence information of the red-eared slider contains 42,165 proteins; the protein sequence information of the pig contains 99,472 proteins; and the protein sequence information of the cattle contains 149,022 proteins.

[0069] There are only 184 proteins of the flower turtle in the protein database (the protein sequence information in the protein database is less than 1,000). The protein information of its genus (Pseudoterrapinna) after removing the proteins of the grass turtle (79,936 proteins) is used as the protein sequence information of the flower turtle.

[0070] According to steps S2 to S4 of Example 1, characteristic peptides among five species were screened and obtained.

[0071] 2. Experimental results

[0072] According to the above method, grass turtle, flower turtle, pig, cattle and red-eared slider were used as the species to be detected, and the characteristic peptide information obtained by screening was shown in Table 1, including grass turtle characteristic peptides such as amino acid sequences shown in SEQ ID NOs: 1 to 14, red-eared slider characteristic peptides such as amino acid sequences shown in SEQ ID NOs: 15 to 20, flower turtle characteristic peptides such as amino acid sequences shown in SEQ ID NOs: 21 to 25, pig characteristic peptides such as amino acid sequences shown in SEQ ID NOs: 26, cattle characteristic peptides such as amino acid sequences shown in SEQ ID NOs: 27 to 31, grass turtle-flower turtle common characteristic peptides such as amino acid sequences shown in SEQ ID NOs: 32 to 35, red-eared slider-flower turtle-pig-cattle common characteristic peptides such as amino acid sequences shown in SEQ ID NOs: 36, and pig-cattle common characteristic peptides such as amino acid sequences shown in SEQ ID NOs: 37 to 45.

[0073] Table 1 Characteristic peptides

[0074]

[0075]

[0076] A total of 18 characteristic peptides of grass turtles, 7 characteristic peptides of red-eared sliders, 10 characteristic peptides of spotted turtles, 11 characteristic peptides of pigs and 15 characteristic peptides of cattle were obtained.

[0077] Example 3 A method for identifying the components of tortoise shell glue

[0078] 1. Detection of peptides in tortoise shell glue

[0079] The tortoise shell glue to be tested was placed in a beaker and covered with deionized water, then transferred to a 100°C water bath. After water bath treatment for 24 hours, it was extracted with n-hexane and the lower liquid was collected. It was lyophilized to obtain tortoise shell glue freeze-dried powder. 100 mg of tortoise shell glue freeze-dried powder was dissolved in 1 mL of water, and 9 mL of trifluoroacetic acid-acetone solution (volume ratio of 1:9) was added. After mixing evenly, it was reacted at -20°C for 12 hours, and the precipitate was collected by centrifugation to obtain the protein to be tested.

[0080] The protein to be tested was dissolved in 500 μL of 100 mM Tris-HCl (pH 8.0) to a protein concentration of 10 mg / mL, and then 100 μL of 100 mM dithiothreitol (DTT) was added and treated in a 59°C water bath for 30 min. Then, 100 μL of 100 mM iodoacetamide (IAA) was added thereto, and the solution was kept in the dark for 30 min to obtain a protein solution; the protein solution was transferred to a 10 kd ultracentrifuge tube and centrifuged for 15 min. The centrifuged protein solution was replaced with 2 mL of 50 mM ammonium bicarbonate solution combined with an ultrafiltration membrane, and then 10 μL of 5 mg / mL trypsin hydrolyzate was added. The solution was placed in a 37°C water bath and heated for 18 h. After heating in a water bath, the solution was concentrated using a freeze concentrator and volatile salts were removed. The solution was then reconstituted with deionized water to a protein concentration of 0.15 mg / mL, and passed through a polyethersulfone filter (PES filter) to obtain a peptide solution to be tested.

[0081] The peptide solution to be tested is combined with ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Orbitrap MS / MS) for detection, specifically: the peptide solution to be tested is adsorbed on a C18 C-trap column, a nano-flow liquid phase system is used to flush the peptide solution to be tested on the C-trap column with a less polar mobile phase into a chromatographic analysis column, and then the solution is detected on a mass spectrometer (including a PRM scanner) to obtain the QE result of the tortoise shell glue to be tested;

[0082] The chromatographic conditions are:

[0083] Chromatographic column: Acclaim PepMap RSLC nanoViper C18 (75 μm × 75 cm, 2 μm, 100A);

[0084] Flow rate: 0.3ul / min;

[0085] Injection volume: 2 μl;

[0086] Mobile phase: Mobile phase A was 0.1% (v / v) formic acid in water, and mobile phase B was 0.1% (v / v) formic acid in water containing 80% (v / v) acetonitrile;

[0087] Nanofluidic elution program: 0-5 min, 5% mobile phase B; 5-25 min, 5%-25% mobile phase B; 25-35 min, 25%-55% mobile phase B; 35-70 min, 55%-80% mobile phase B; 70-90 min, 80% mobile phase B; 90-100 min, 80%-5% mobile phase B; 100-130 min, 5% mobile phase B.

[0088] Mass spectrometry conditions:

[0089] Mass spectrometry: THERMO Q-Exative-orbitrap MS equipped with a nanoelectrospray ionization (NSI) source;

[0090] Spray voltage: positive ion mode;

[0091] Ion transfer tube temperature: 250°C;

[0092] Sheath gas flow rate: 5arb;

[0093] Auxiliary gas flow rate: 2arb;

[0094] S-lens voltage: 50v;

[0095] Heating temperature: 250℃;

[0096] Scan range: 150-2000 m / z;

[0097] Full scan resolution of mass spectrometry: R = 70000;

[0098] Automatic gain control for Full MS with target ion number in C-Trap (AGC target): 3e 6 ;

[0099] Maximum injection time (Maximum IT): 50ms;

[0100] Data-dependent secondary ion full scan resolution: R = 17500;

[0101] AGC target: 2e 5 ;

[0102] Maximum injection time: 50ms;

[0103] Normalized collision energy: 28 eV;

[0104] The first 10 precursor ions were fed into the secondary mass spectrometer;

[0105] Dynamic exclusion: 10s.

[0106] The chromatographic conditions in the PRM instrument scanning parameters were consistent with the above chromatographic conditions;

[0107] Mass spectrometry conditions in PRM instrument scanning parameters:

[0108] Data dependent secondary ion full scan resolution: Default charge state: 2;

[0109] Full scan resolution of mass spectrometry: R = 17500;

[0110] Automatic gain control for Full MS with target ion number in C-Trap (AGC target): 1e 5 ;

[0111] Maximum injection time: 50ms;

[0112] Normalized collision energy: 28 eV.

[0113] The QE results of the tortoise shell glue to be tested were imported into Proteome Discoverer software (Thermo Fisher Scientific, Waltham, MA, USA), and the protein databases of Chinese tortoise, red-eared slider, red-eared slider, pig, and cattle were recorded. The QE results of the tortoise shell glue to be tested were analyzed to obtain the peptide information of the tortoise shell glue to be tested;

[0114] When using Proteome Discoverer software for protein and peptide identification, the parameters were set as follows: trypsin digestion, no missed cleavage sites allowed; peptide precursor ion error tolerance of 10 ppm, fragment ion error tolerance of 0.02 Da; high confidence (FDR < 1%), coverage > 50%; ion strength > 1 × 10 7 ; Peptide length>4; Other parameters are software default parameters.

[0115] 2. Identification of the components in the tortoise shell glue to be tested

[0116] The peptide information of the tortoise shell glue to be tested obtained in step 1 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 1 to 14, indicating that the tortoise shell glue to be tested contains tortoise-derived substances;

[0117] The peptide information of the tortoise shell glue to be tested obtained in step 1 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 15 to 20, indicating that the tortoise shell glue to be tested contains a substance derived from red-eared turtles;

[0118] The peptide information of the tortoise shell glue to be tested obtained in step 1 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 21 to 25, indicating that the tortoise shell glue to be tested contains tortoise-derived substances;

[0119] The peptide information of the tortoise shell glue to be tested obtained in step 1 contains a characteristic peptide with an amino acid sequence as shown in SEQ ID NO: 26, indicating that the tortoise shell glue to be tested contains porcine-derived substances;

[0120] The peptide information of the tortoise shell glue to be tested obtained in step 1 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 27 to 31, indicating that the tortoise shell glue to be tested contains bovine-derived substances;

[0121] The peptide information of the tortoise shell glue to be tested obtained in step 1 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 32 to 35, and the tortoise shell glue to be tested contains one or both of the tortoise-derived substance and the tortoise-derived substance;

[0122] The peptide information of the tortoise shell glue to be tested obtained in step 1 contains a characteristic peptide with an amino acid sequence as shown in SEQ ID NO: 36, and the tortoise shell glue to be tested contains one or more of a red-eared turtle-derived substance, a turtle-derived substance, a porcine-derived substance, and a bovine-derived substance;

[0123] The peptide information of the tortoise shell glue to be tested obtained in step 1 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 37 to 45, and the tortoise shell glue to be tested contains one or both of porcine and bovine substances.

[0124] Example 4: Verification of a method for identifying components of tortoise shell glue

[0125] 1. Experimental methods

[0126] Ten different tortoise shell glue products commonly found on the market were used as test objects to identify the components of tortoise shell glue; the ten different tortoise shell glue products included Jinqian brand tortoise jelly powder (sample 1), Shuangqian brand tortoise jelly powder (sample 2), Shuanghengbao tortoise jelly powder (sample 3), Shenghetang tortoise jelly powder (sample 4), Yufeng tortoise jelly powder (sample 5), Guiyishoushi tortoise jelly (sample 6), Luyou Pharmaceutical tortoise shell glue (sample 7), Guinajian tortoise shell glue (sample 8), Dong'e Ejiao tortoise shell glue (sample 9) and Runjia tortoise shell glue (sample 10); among them, samples 1 to 5 were tortoise jelly powders, and samples 6 to 10 were tortoise jelly pastes.

[0127] Pretreatment of guilinggao powder: 1 g of guilinggao powder was placed in a centrifuge tube, 5 mL of Tris-HCl buffer was added and mixed thoroughly. After the guilinggao powder was dissolved, 5 mL of Tris-phenol extraction reagent (80%, v / v) was added thereto and mixed thoroughly at 25°C. The mixture was then centrifuged at 4°C, 4000×g for 10 min. The upper layer of liquid was transferred to a new centrifuge tube, 20 mL of cold methanol containing 0.1 mol / L ammonium acetate was added, the mixture was mixed thoroughly, and the mixture was allowed to stand for 12 h. The precipitate was collected by centrifugation and rinsed three times with acetone. After rinsing, the precipitate was freeze-dried to obtain lyophilized guilinggao powder.

[0128] The difference between the pretreatment of tortoise jelly paste and the pretreatment of tortoise jelly powder is that 1g of tortoise jelly powder is changed to 5g of tortoise jelly paste, and the other treatments remain unchanged to obtain tortoise shell glue freeze-dried powder.

[0129] The freeze-dried powder of tortoise shell glue was used to identify the components of tortoise shell glue according to the method shown in Example 3.

[0130] 2. Experimental results

[0131] The composition identification results of 10 different tortoise shell glue products are shown in Table 2.

[0132] Table 2 Component identification results of tortoise shell glue products

[0133]

[0134]

[0135] The results showed that the peptide segment of sample 7 contained a pig-cow common characteristic peptide, indicating that sample 7 contained one or both of pig-derived substances and cow-derived substances; the peptide segment of sample 8 contained a red-eared slider characteristic peptide, indicating that its ingredients contained red-eared slider derived substances.

[0136] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for screening characteristic peptides between different species, using at least two different species as the species to be detected, characterized in that: The following steps are involved: S1. Obtain protein sequence information of the species to be detected from the protein database; When the number of protein sequence information of the species to be detected in the protein database is less than 1000, the protein sequence information of the species to be detected in the protein database after removing the species other than the species to be detected from the genus to which the species to be detected belongs is used as the protein sequence information of the species to be detected; when the number of protein sequence information of the species to be detected in the protein database is less than 1000, the protein sequence information of the species to be detected in the protein database is used as the protein sequence information of the species to be detected; S2. Performing theoretical enzyme digestion on the protein sequence information of the species to be detected obtained in step S1 to obtain theoretical enzyme-digested peptides of the species to be detected, and recording the source protein of the theoretical enzyme-digested peptides of the species to be detected; S3. Screening the theoretical enzyme-cleaved peptides of the species to be detected obtained in step S2 to obtain differential peptides of the species to be detected; The screening specifically includes: removing peptides with a length of less than 4 aa or greater than 20 aa in the theoretical enzyme-cleaved peptides of the species to be detected, removing peptides that appear repeatedly in the theoretical enzyme-cleaved peptides of different species to be detected, and deduplicating peptides that appear repeatedly in the theoretical enzyme-cleaved peptides of the species to be detected; S4. Selecting peptides that meet the selection criteria from the differential peptides of the species to be detected obtained in step S3 as characteristic peptides of the species to be detected; The selection conditions are: the differential peptide segment of the species to be detected does not exist in the protein sequence information of the species to be detected other than the species to be detected, the source protein of the differential peptide segment of the species to be detected exists in the protein of the species to be detected other than the species to be detected, and the sequence identity between the differential peptide segment of the species to be detected and the peptide segment in the species to be detected other than the species to be detected is not less than 50%; The peptides in the species to be detected other than the species to be detected are derived from the same protein as the differential peptides in the species to be detected, and their positions on the protein are offset by no more than 20 aa from the positions of the differential peptides in the species to be detected on the protein.

2. The screening method according to claim 1, wherein The protein database in step S1 is the NCBI database, the Uniprot database and / or the PDB database.

3. The screening method according to claim 1, wherein The theoretical enzymatic digestion in step S2 is theoretical enzymatic digestion by trypsin and / or theoretical enzymatic digestion by pepsin.

4. The screening method according to claim 3, wherein The theoretical enzymatic digestion in step S2 is a theoretical enzymatic digestion using trypsin.

5. Use of the screening method according to any one of claims 1 to 4 in screening characteristic peptides between different species.

6. A characteristic peptide combination for identifying tortoise shell glue components, characterized in that: It includes characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 1 to 45.

7. Use of the characteristic peptide combination according to claim 6 in identifying components of tortoise shell glue.

8. The use according to claim 7, characterized in that The identification of tortoise shell glue components is to identify whether there are tortoise-derived substances, red-eared slider-derived substances, spotted turtle-derived substances, pig-derived substances and / or cattle-derived substances in the tortoise shell glue.

9. Use of the characteristic peptide combination according to claim 6 as a quantitative marker in the quantitative detection of tortoise shell glue components.

10. A method for identifying the components of tortoise shell glue, characterized in that: The following steps are involved: S11. Using the tortoise shell glue to be tested as a sample, ultra-performance liquid chromatography-high-resolution mass spectrometry is used to obtain mass spectrometry results, and based on the mass spectrometry results, peptide information of the tortoise shell glue to be tested is obtained; S12. The peptide information of the tortoise shell glue to be detected obtained in step S11 is combined with the characteristic peptide combination of claim 6 to obtain the identification result of the tortoise shell glue to be detected; The identification is specifically performed in combination with the characteristic peptide combination of claim 6: the peptide information of the tortoise shell glue to be detected obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 1 to 14, and the tortoise shell glue to be detected contains tortoise-derived substances; The peptide information of the tortoise shell glue to be tested obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 15 to 20, indicating that the tortoise shell glue to be tested contains a substance derived from red-eared turtles; The peptide information of the tortoise shell glue to be tested obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 21 to 25, indicating that the tortoise shell glue to be tested contains tortoise-derived substances; The peptide information of the tortoise shell glue to be tested obtained in step S11 contains a characteristic peptide with an amino acid sequence as shown in SEQ ID NO: 26, indicating that the tortoise shell glue to be tested contains porcine-derived substances; The peptide information of the tortoise shell glue to be tested obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 27 to 31, indicating that the tortoise shell glue to be tested contains bovine-derived substances; The peptide information of the tortoise shell glue to be tested obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 32 to 35, and the tortoise shell glue to be tested contains tortoise-derived substances and / or tortoise-derived substances; The peptide information of the tortoise shell glue to be tested obtained in step S11 contains a characteristic peptide with an amino acid sequence as shown in SEQ ID NO: 36, and the tortoise shell glue to be tested contains a red-eared turtle-derived substance, a turtle-derived substance, a porcine-derived substance, and / or a bovine-derived substance; The peptide information of the tortoise shell glue to be detected obtained in step S11 contains characteristic peptides with amino acid sequences as shown in SEQ ID NOs: 37 to 45, and the tortoise shell glue to be detected contains porcine-derived substances and / or bovine-derived substances.

Images