Bovine type I collagen terminal end peptide marker polypeptide as well as preparation method and application thereof

Abstract

The invention provides a bovine type I collagen terminal end peptide characteristic polypeptide, the bovine type I collagen terminal end peptide characteristic polypeptide comprises an N terminal end peptide characteristic polypeptide and a C terminal end peptide characteristic polypeptide, the amino acid sequence of the N terminal end peptide characteristic polypeptide is Q * LSYGYDE, and the amino acid sequence of the C terminal end peptide is LSFLPQPPQE. The invention also provides application of the bovine type I collagen terminal peptide marker polypeptide in detection of N-terminal peptide and C-terminal peptide of bovine type I collagen. The invention also provides a preparation method of a collagen enzymolysis product containing the bovine type I collagen terminal peptide characteristic polypeptide. The bovine type I collagen tail end terminal peptide marker polypeptide provided by the invention is a special terminal peptide at the tail end of bovine type I collagen. The terminal peptide marker polypeptide is used as a marker and can be used for quickly identifying the bovine type I collagen terminal peptide and accurately and quantitatively analyzing the bovine type I collagen terminal peptide. The qualitative and quantitative detection methods are simple to operate and high in sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of collagen polypeptides, in particular to a bovine type I collagen terminal telopeptide characteristic polypeptide and application of the bovine type I collagen terminal telopeptide characteristic polypeptide in qualitative and quantitative analysis of bovine type I collagen telopeptide. Technical Background

[0002] Bovine type I collagen is prepared from bovine Achilles tendon, skin, bone, and other raw materials through enzymatic extraction. The bovine type I collagen molecule consists of a triple-helical core region and terminal telopeptides. The core region is a triple-helical structure formed by two α1 chains and one α2 chain, each peptide chain consisting of a repeating sequence of Gly-XY. The terminal telopeptides are divided into N-terminal and C-terminal non-helical regions. Bovine type I collagen has excellent biocompatibility, biodegradability, and self-assembly properties, which have led to its widespread application in biomaterials, medical devices, and other fields. However, the immunogenicity of animal-derived collagen limits its effectiveness. The removal rate of telopeptides is the main factor affecting its immunogenicity. Therefore, the establishment of a high-precision and high-sensitivity telopeptide detection method is of great significance for the quality evaluation, process improvement, and immunogenicity assessment of bovine type I collagen products.

[0003] Currently, commonly used methods for detecting collagen telopeptides include enzyme-linked immunosorbent assay (ELISA), liquid chromatography (HPLC), and chemiluminescent immunoassay. The ELISA method utilizes specific reverse-phase reactions between antibodies and antigens for qualitative and quantitative analysis. However, this method is cumbersome and requires multiple, complex steps to complete the assay, which is time-consuming and labor-intensive, resulting in high testing costs. Furthermore, nonspecific adsorption can lead to false-positive results, compromising the accuracy and reliability of the assay. Liquid chromatography (HPLC) has limitations in determining complete telopeptide removal by measuring the tyrosine content in bovine type I collagen. Because the α2 chain of bovine type I collagen contains tyrosine, the measured tyrosine content cannot be fully equated with the telopeptide content. This method is subject to certain errors and cannot accurately determine complete telopeptide removal or precisely quantify the amount of residual telopeptide in collagen. For example, chemiluminescent immunoassays, such as magnetic microparticle chemiluminescent immunoassays, require re-screening of capture and detection antibodies for different types of collagen telopeptides. This requires extensive preliminary research, including complex processes such as antigen preparation and antibody screening. Moreover, this method has poor universality and cannot be widely used in the detection of various types of collagen telopeptides, which limits its promotion in practical applications.

[0004] In summary, these existing detection methods have technical problems that need to be solved, such as high cost, time-consuming and labor-intensive, or inaccurate detection. Summary of the Invention

[0005] In order to solve at least one of the above problems, the present invention provides a bovine type I collagen terminal telopeptide characteristic polypeptide and its preparation method, and uses the bovine type I collagen terminal telopeptide characteristic polypeptide to perform qualitative and quantitative detection of bovine type I collagen terminal telopeptide.

[0006] On the one hand, the present invention provides a bovine type I collagen terminal telopeptide characteristic polypeptide, characterized in that the bovine type I collagen terminal telopeptide characteristic polypeptide includes an N-terminal telopeptide characteristic polypeptide and a C-terminal telopeptide characteristic polypeptide, the amino acid sequence of the N-terminal telopeptide characteristic polypeptide is Q*LSYGYDE (Q* is pyroglutamic acid), and the amino acid sequence of the C-terminal telopeptide is LSFLPQPPQE.

[0007] On the other hand, the present invention provides a use of the above-mentioned bovine type I collagen terminal telopeptide characteristic polypeptide in detecting bovine type I collagen N-terminal telopeptide and C-terminal telopeptide.

[0008] On the other hand, the present invention also provides a method for preparing a collagenase hydrolysate containing the bovine type I collagen terminal telopeptide characteristic polypeptide, characterized in that the preparation method comprises the following steps: 1) thermally denaturing type I collagen in a boiling water bath for 20-40 minutes to depolymerize the triple helical structure;

[0009] 2) Enzymatic digestion of bovine type I collagen using Glu-C for 16-20 h at a temperature of 35-40°C;

[0010] 3) analyzing the obtained enzymatic hydrolysis products using a mass spectrometer to obtain a total ion current of the bovine type I collagen enzymatic hydrolysis products;

[0011] 4) importing the sequence of bovine type I collagen into BioFinder software, and aligning the obtained spectrum with the sequence of bovine type I collagen using BioFinder software;

[0012] 5) The comparison results showed that the enzymatic hydrolysis product of bovine type I collagen contained an ion of m / z 1155.58 in the primary mass spectrum, with a molecular weight of 1154.60 Da, which was consistent with the theoretical molecular weight of the polypeptide LSFLPQPPQE; in the secondary mass spectrum of m / z 1155.58, its fragment ion was highly matched with the theoretical fragment ion of the polypeptide LSFLPQPPQE, indicating that the polypeptide LSFLPQPPQE only existed in the C-terminal telopeptide of bovine type I collagen; the enzymatic hydrolysis product of bovine type I collagen contained an ion of m / z 957.38 in the primary mass spectrum, with a molecular weight of 956.4, which was consistent with the theoretical molecular weight of the polypeptide Q*LSYGYDE; in the secondary mass spectrum of m / z 957.38, its fragment ion was highly matched with the theoretical fragment ion of the polypeptide Q*LSYGYDE, indicating that the polypeptide Q*LSYGYDE only existed in the N-terminal telopeptide of bovine type I collagen.

[0013] The present invention provides a unique bovine type I collagen terminal telopeptide-specific polypeptide. Using this terminal telopeptide-specific polypeptide as a marker, it can be used for rapid identification of bovine type I collagen telopeptides and accurate quantitative analysis. These qualitative and quantitative detection methods are simple to operate and highly sensitive, enabling rapid and accurate analysis of residual bovine type I collagen telopeptides and product quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the total ion current of the enzymatic hydrolysis product of bovine type I collagen Glu-C;

[0015] Figure 2 This is the extracted ion chromatogram of m / z 1155.60352 in the enzymatic hydrolysis product of bovine type I collagen Glu-C;

[0016] Figure 3 This is the primary mass spectrum of m / z 1155.60352 in the enzymatic hydrolysis product of bovine type I collagen Glu-C;

[0017] Figure 4 This is the secondary mass spectrum of m / z 1155.60352 in the enzymatic hydrolysis product of bovine type I collagen Glu-C;

[0018] Figure 5 This is the extracted ion chromatogram of m / z 957.38892 in the enzymatic hydrolysis product of bovine type I collagen Glu-C;

[0019] Figure 6 This is the primary mass spectrum of m / z 957.38892 in the enzymatic hydrolysis product of bovine type I collagen Glu-C;

[0020] Figure 7 This is the secondary mass spectrum of m / z 957.38892 in the enzymatic hydrolysis product of bovine type I collagen Glu-C;

[0021] Figure 8 This is the primary mass spectrum of the characteristic peptide of the N-terminal telopeptide of bovine type I collagen;

[0022] Figure 9 This is the secondary mass spectrometry of the characteristic peptide of the N-terminal telopeptide of bovine type I collagen;

[0023] Figure 10 The spectra of the standard solutions of bovine type I collagen N-terminal telopeptide characteristic polypeptides at different concentrations;

[0024] Figure 11 This is the primary mass spectrum of the characteristic peptide of the C-terminal telopeptide of bovine type I collagen;

[0025] Figure 12This is the secondary mass spectrometry of the characteristic peptide of the C-terminal telopeptide of bovine type I collagen;

[0026] Figure 13 The spectra of different concentrations of bovine type I collagen C-terminal telopeptide characteristic polypeptide standard solutions;

[0027] Figure 14 This is the N-terminal telopeptide spectrum of bovine type I collagen sample;

[0028] Figure 15 This is the C-terminal telopeptide spectrum of bovine type I collagen sample. Specific embodiments

[0029] The present invention provides a bovine type I collagen terminal telopeptide characteristic polypeptide, which is characterized in that the bovine type I collagen terminal telopeptide characteristic polypeptide comprises an N-terminal telopeptide characteristic polypeptide and a C-terminal telopeptide characteristic polypeptide, the amino acid sequence of the N-terminal telopeptide characteristic polypeptide is Q*LSYGYDE (Q* is pyroglutamic acid), and the amino acid sequence of the C-terminal telopeptide is LSFLPQPPQE.

[0030] The present invention also provides the use of the bovine type I collagen terminal telopeptide characteristic polypeptide in detecting the N-terminal telopeptide and C-terminal telopeptide of bovine type I collagen. For example, the use of the polypeptide can be used to detect the presence and concentration of the N-terminal telopeptide and / or C-terminal telopeptide in bovine type I collagen raw materials and medical device products extracted from bovine tendon, skin, bone, and other tissues.

[0031] In a preferred embodiment, the detection method is liquid chromatography-tandem mass spectrometry.

[0032] In another preferred embodiment, the detection method is qualitative detection, and the liquid chromatography-tandem mass spectrometry detection method comprises the following steps:

[0033] 1) preparing the N-terminal telopeptide characteristic polypeptide and the C-terminal telopeptide characteristic polypeptide into solution A and solution B of certain concentrations respectively;

[0034] 2) Injecting the solution A, the solution B and the sample to be tested into a liquid chromatography-mass spectrometer respectively;

[0035] 3) If the retention time of the daughter ion in the sample to be tested is the same as the retention time of the daughter ion of the N-terminal telopeptide characteristic polypeptide and / or the C-terminal telopeptide characteristic polypeptide, then the N-terminal telopeptide characteristic polypeptide and / or the C-terminal telopeptide characteristic polypeptide is present in the sample to be tested.

[0036] In a preferred embodiment, the daughter ion of the N-terminal telopeptide characteristic polypeptide is m / z 957.38→532.03,695.19, and the daughter ion of the C-terminal telopeptide characteristic polypeptide is m / z 1155.58→470.03,695.23, as the detection ion to detect the retention time.

[0037] In a preferred embodiment, the detection method is quantitative detection, and the liquid chromatography-tandem mass spectrometry detection method comprises the following steps:

[0038] A. Developing a concentration standard curve: Prepare the N-terminal telopeptide characteristic polypeptide and the C-terminal telopeptide characteristic polypeptide into standard solutions of different concentrations, and perform liquid chromatography-tandem mass spectrometry detection to draw a concentration standard curve. 2 The standard curve of concentration and peak area is greater than 0.99;

[0039] B. Detecting the sample by liquid chromatography-tandem mass spectrometry;

[0040] C. Calculate the C-terminal telopeptide content of bovine type I collagen in the sample according to formula (1), and calculate the N-terminal telopeptide content of bovine type I collagen in the sample according to formula (2):

[0041]

[0042] Where: W C : C-terminal telopeptide content of bovine type I collagen in the sample to be tested, μg / mg; X C : concentration of bovine type I collagen C-terminal telopeptide characteristic polypeptide, μg / mL; V: final volume of the sample to be tested, mL; M C : Molecular weight of bovine type I collagen C-terminal telopeptide, u; M CP : Molecular weight of the characteristic polypeptide of bovine type I collagen C-terminal telopeptide, u; m: Mass of the sample to be tested, mg; n C : The number of repetitions of the C-terminal telopeptide characteristic polypeptide in bovine type I collagen;

[0043]

[0044] Where: W N : N-terminal telopeptide content of bovine type I collagen in the sample to be tested, μg / mg; X N : concentration of bovine type I collagen N-terminal telopeptide characteristic polypeptide, μg / mL; V: final volume of the sample to be tested, mL; M N : molecular weight of bovine type I collagen N-terminal telopeptide, u; M NP : molecular weight of bovine type I collagen N-terminal telopeptide characteristic polypeptide, u; m: mass of the sample to be tested, mg; n N The number of repeats of the N-terminal telopeptide signature polypeptide in bovine type I collagen.

[0045] When developing a concentration standard curve, a series of concentrations of nanograms per milliliter and micrograms per milliliter can be prepared as needed, as long as R 2 A concentration vs. peak area standard curve with a value greater than 0.99 is sufficient. For example, a concentration standard curve can be prepared as follows: Accurately weigh 10 mg of bovine type I collagen N-terminal characteristic peptide standard and C-terminal characteristic peptide standard, place them in a 10 mL volumetric flask, dissolve them in 0.1 mol / L Tris-HCl (pH 8.0), and dilute to volume to prepare the terminal peptide characteristic peptide standard stock solution. The bovine type I collagen N-terminal characteristic polypeptide stock solution was diluted with 0.1 mol / L Tris-HCl (pH 8.0) solution into a series of standard working solutions with concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL; the bovine type I collagen C-terminal characteristic polypeptide stock solution was diluted using the same method into a series of standard working solutions with concentrations of 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL.

[0046] In a preferred embodiment, the daughter ion of the N-terminal telopeptide characteristic polypeptide is m / z 957.38→532.03,695.19, and the daughter ion of the C-terminal telopeptide characteristic polypeptide is m / z 1155.58→470.03,695.23, as the detection ion to detect the retention time.

[0047] A buffer solution can be used to prepare the N-terminal telopeptide characteristic polypeptide solution or the C-terminal telopeptide characteristic polypeptide solution. The buffer solution can be a buffer commonly used in the art, for example, Tris-HCl, ammonium bicarbonate buffer, etc. However, in a preferred embodiment, a Tris-HCl solution (for example, 0.1 mol / L Tris-HCl (pH 8.0)) is used to prepare the N-terminal telopeptide characteristic polypeptide solution or the C-terminal telopeptide characteristic polypeptide solution.

[0048] In a preferred embodiment, the mass spectrometry analysis of the N-terminal telopeptide characteristic polypeptide and the C-terminal telopeptide characteristic polypeptide are both performed by electrospray ionization, positive ion mode, and multiple reaction monitoring.

[0049] In a preferred embodiment, the liquid chromatography parameters are: Zorbax C18 column; mobile phase: A is water containing 0.1% formic acid, B is 60% acetonitrile in water containing 0.1% formic acid (V / V); gradient elution: 0-0.5 min (5% B); 0.5-2 min (5%-45% B), 2-3 min (45%-90% B), 3-4.5 min (90%-100% B), 4.5-7 min (100% B), 7-7.1 (100%-5% B); 7.1-10 min (5% B); flow rate 0.2 ml / min; column temperature: 30°C; sample load: 5.0 μL.

[0050] In a preferred embodiment, the mass spectrometry analysis conditions are: ion source spray voltage 3.5 kV, capillary temperature 320 ° C, evaporation temperature 350 ° C, sheath gas flow rate 19.5 mL / min, auxiliary flow rate 0.37 bar, positive ion mode scanning, SRM monitoring mode: the N-terminal terminal peptide characteristic polypeptide m / z 957.38 single charge → 532.03 (Tube Lens = 140, CE = 28), the C-terminal terminal peptide characteristic polypeptide m / z 1155.58 single charge → 470.03 (Tube Lens = 149, CE = 43).

[0051] The present invention also provides a method for preparing the bovine type I collagen terminal telopeptide characteristic polypeptide, characterized in that the preparation method comprises the following steps: 1) thermally denaturing the type I collagen in a boiling water bath for 20-40 minutes to depolymerize the triple helical structure;

[0052] 2) Enzymatic digestion of bovine type I collagen using Glu-C for 16-20 h at a temperature of 35-40°C;

[0053] 3) analyzing the obtained enzymatic hydrolysis products using a mass spectrometer to obtain a total ion current of the bovine type I collagen enzymatic hydrolysis products;

[0054] 4) importing the sequence of bovine type I collagen into BioFinder software, and aligning the obtained spectrum with the sequence of bovine type I collagen using BioFinder software;

[0055] 5) The comparison results showed that the enzymatic hydrolysis product of bovine type I collagen contained an ion of m / z 1155.58 in the primary mass spectrum, with a molecular weight of 1154.60 Da, which was consistent with the theoretical molecular weight of the polypeptide LSFLPQPPQE; in the secondary mass spectrum of m / z 1155.58, its fragment ion was highly matched with the theoretical fragment ion of the polypeptide LSFLPQPPQE, indicating that the polypeptide LSFLPQPPQE only existed in the C-terminal telopeptide of bovine type I collagen; the enzymatic hydrolysis product of bovine type I collagen contained an ion of m / z 957.38 in the primary mass spectrum, with a molecular weight of 956.4, which was consistent with the theoretical molecular weight of the polypeptide Q*LSYGYDE; in the secondary mass spectrum of m / z 957.38, its fragment ion was highly matched with the theoretical fragment ion of the polypeptide Q*LSYGYDE, indicating that the polypeptide Q*LSYGYDE only existed in the N-terminal telopeptide of bovine type I collagen.

[0056] In the present application, it should be noted that the number after the decimal point of the parameter m / z value may vary slightly due to the different resolutions of the detection instrument. The number after the decimal point of the parameter m / z value in the claims should be considered to be equivalent to the corresponding value specified in the claims.

[0057] Example 1: Preparation method of collagenase hydrolyzed product containing bovine type I collagen terminal peptide characteristic polypeptide

[0058] A telopeptide-containing bovine type I collagen acellular matrix was prepared at a concentration of 0.5 mg / mL and heat-denatured in a boiling water bath for 30 minutes to disaggregate the triple helical structure. The bovine type I collagen was then enzymatically digested with Glu-C for 18 hours at 37°C. The resulting enzymatic products were analyzed using a high-resolution mass spectrometer (Orbitrap Exploris 480). Figure 1 This is the total ion current of the enzymatic hydrolysis product of bovine type I collagen Glu-C. The sequence of bovine type I collagen was imported into BioFinder software, and the resulting spectrum was compared with the sequence of bovine type I collagen using BioFinder software. The comparison results show that the ion m / z 1155.60352 exists in the enzymatic hydrolysis product of bovine type I collagen. Figure 2 The primary mass spectrum of this ion is shown in Figure 1. The isotopic peak of this ion indicates that it is singly charged. Therefore, the molecular weight of the ion m / z 1155.60352 is 1154.60352 Da, which is consistent with the theoretical molecular weight of the peptide LSFLPQPPQE. Figure 3The secondary mass spectrum of m / z 1155.60352 has a fragment ion that closely matches the theoretical fragment ion of the peptide LSFLPQPPQE. A search on the Uniprot website revealed that this peptide is only found in the C-terminal telopeptide of bovine type I collagen. The primary mass spectrum of the enzymatic hydrolysis product of bovine type I collagen contains an ion at m / z 957.38, with a molecular weight of 956.4, which is consistent with the theoretical molecular weight of the peptide Q*LSYGYDE (Q* is pyroglutamic acid). Figure 4 ), its fragment ions are highly consistent with the theoretical fragment ions of the peptide Q*LSYGYDE, which is present only in the N-terminal telopeptide of bovine type I collagen. Its primary and secondary mass spectra are shown in Figure 5-Figure 7 Therefore, the peptides LSFLPQPPQE and Q*LSYGYDE can be the characteristic peptides of the N-terminal and C-terminal telopeptides of bovine type I collagen, respectively.

[0059] Example 2

[0060] Detection of Telopeptide Content in Bovine Type I Collagen Samples

[0061] 1 Experimental materials and instruments

[0062] 1.1 Experimental Reagents

[0063] The synthetic peptide standards LSFLPQPPQE and Q*LSYGYDE were provided by Suzhou Qiangyao Biotechnology Co., Ltd.; bovine type I collagen sample; Glu-C enzyme (sequence pure) were purchased from Promega Corporation, USA; chromatographic grade acetonitrile was purchased from Merck, USA; chromatographic grade formic acid was purchased from Thermo Fisher Scientific, USA; other reagents were all commercially available and of analytical grade.

[0064] 1.2 Experimental instruments

[0065] The analytical balance (AL104) was from Mettler Toledo; the centrifuge (PK165) was from Hunan Pingke Scientific Instrument Co., Ltd.; the constant temperature water bath shaker (SHA-BA) was from Changzhou Ronghua Instrument Manufacturing Co., Ltd.; the liquid chromatograph (U 3000) and triple quadrupole liquid chromatography-mass spectrometer (Quantum ACCESS MAX) were both from Thermo Fisher Scientific, USA; and the data processing software was Xcalibur 3.1.

[0066] 2 Experimental methods

[0067] 2.1 Preparation of LSFLPQPPQE and Q*LSYGYDE Standard Solutions

[0068] Accurately weigh 10 mg of bovine type I collagen terminal telopeptide characteristic peptide standards (LSFLPQPPQE and Q*LSYGYDE standards) into a 10 mL volumetric flask and dissolve and dilute to volume with 0.1 mol / L Tris-HCl (pH 8.0) to prepare the characteristic peptide standard stock solutions. Dilute the bovine type I collagen N-terminal telopeptide characteristic peptide stock solution with 0.1 mol / L Tris-HCl (pH 8.0) to prepare a series of standard working solutions at concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. Use the same method to dilute the C-terminal telopeptide characteristic peptide to prepare a series of standard working solutions at concentrations of 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 5 μg / mL.

[0069] 2.2 Bovine type I collagen sample processing method

[0070] Accurately weigh 5.0 mg of bovine type I collagen sample and place it in a 15 mL centrifuge tube. Add 10 mL of 0.1 mol / L Tris-HCl (pH 8.0) solution and heat denature in a boiling water bath for 30 min to fully dissolve it. Take 0.5 mL of the dissolved bovine type I collagen solution and mix it with 10 μg of Glu-C enzyme. Place it in a 37°C constant temperature water bath shaker and react for 18 h. Centrifuge (10,000 r / min, 10 min) and take the supernatant for analysis.

[0071] 2.3 Quantitative analysis

[0072] 2.3.1 Liquid chromatography conditions

[0073] Chromatographic column: Zorbax C18 column (2.1 mm × 150 mm, 5 μm); mobile phase: A: water (0.1% formic acid), B: 60% acetonitrile in water (v / v, 0.1% formic acid); gradient elution: 0-0.5 min (5% B); 0.5-2 min (5%-45% B); 2-3 min (45%-90% B); 3-4.5 min (90%-100% B); 4.5-7 min (100% B); 7-7.1 (100%-5% B); 7.1-10 min (5% B); flow rate: 0.2 mL / min; column temperature: 30°C; sample load: 5.0 μL.

[0074] 2.3.2 Mass spectrometry conditions

[0075] The ion source spray voltage was 3.5 kV, the capillary temperature was 320°C, the evaporation temperature was 350°C, the sheath gas flow rate was 19.8 mL / min, the auxiliary flow rate was 0.55 bar, the positive ion mode was scanned, and the SRM monitoring mode was used: N-terminal telopeptide characteristic polypeptide m / z 957.38 (singly charged) → 532.03 (Tube Lens = 140, CE = 28), C-terminal telopeptide characteristic polypeptide m / z 1155.58 (singly charged) → 470.03 (Tube Lens = 149, CE = 43).

[0076] 2.4 Data Processing

[0077] The C-terminal telopeptide content of bovine type I collagen in the sample was calculated according to formula (1), and the N-terminal telopeptide content of bovine type I collagen in the sample was calculated according to formula (2):

[0078]

[0079] Where: W C : C-terminal telopeptide content of bovine type I collagen in the sample to be tested, μg / mg; X C : concentration of bovine type I collagen C-terminal telopeptide characteristic polypeptide, μg / mL; V: final volume of the sample to be tested, mL; M C : Molecular weight of bovine type I collagen C-terminal telopeptide, u; M CP : Molecular weight of the characteristic polypeptide of bovine type I collagen C-terminal telopeptide, u; m: Mass of the sample to be tested, mg; n C : The number of repetitions of the C-terminal telopeptide characteristic polypeptide in bovine type I collagen;

[0080]

[0081] Where: W N : N-terminal telopeptide content of bovine type I collagen in the sample to be tested, μg / mg; X N : concentration of bovine type I collagen N-terminal telopeptide characteristic polypeptide, μg / mL; V: final volume of the sample to be tested, mL; M N : Molecular weight of bovine type I collagen N-terminal telopeptide, u; M NP : molecular weight of bovine type I collagen N-terminal telopeptide characteristic polypeptide, u; m: mass of the sample to be tested, mg; n N The number of repeats of the N-terminal telopeptide signature polypeptide in bovine type I collagen.

[0082] 3 Experimental Results

[0083] 3.1 Methodological validation

[0084] The chromatograms of N-terminal end peptide characteristic polypeptide standard solutions with different concentrations are shown in Figure 10(A, B, C, D, E, and F in the figure are N-terminal terminal peptide characteristic polypeptides with concentrations of 0.5μg / mL, 1μg / mL, 2μg / mL, 5μg / mL, 10μg / mL, and 20μg / mL, respectively). The secondary mass spectrum of the polypeptide is shown in Figure 9 The linear regression equation is y=91343.7+3.39108*10 6 x, R 2 =0.9993. The linear correlation coefficient was greater than 0.99, indicating good linearity for the peptide. During the methodological investigation, the RSD value for the precision experiment was 4.47%, demonstrating good instrument precision; the RSD value for the repeatability experiment was 3.29%, indicating good repeatability of the method. The spike recovery rates at different spiking levels ranged from 89% to 105%, meeting the spike recovery requirements and demonstrating accurate detection results. The limits of quantification and detection for the characteristic peptide were 25 ng / mL and 12 ng / mL, respectively.

[0085] The chromatograms of the C-terminal end peptide characteristic polypeptide standard solutions with different concentrations are shown in Figure 13 (In the figure, A, B, C, D, E, F, G, and H are C-terminal end peptide characteristic polypeptides with concentrations of 0.05μg / mL, 0.1μg / mL, 0.2μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, 5μg / mL, and 10μg / mL, respectively). The secondary mass spectrum of the polypeptide is shown in Figure 12 The linear regression equation is y = -264935 + 2.23013 * 10 7 x, R 2 The linear correlation coefficient was greater than 0.99, indicating good linearity for the peptide. During the methodological investigation, the RSD value for the precision experiment was 3.99%, indicating good instrument precision; the RSD value for the repeatability experiment was 3.06%, indicating good repeatability of the method. The spike recovery rates at different spiking levels ranged from 93% to 101%, meeting the spike recovery requirements and demonstrating accurate detection results. The limits of quantification and detection for the characteristic peptide were 5 ng / mL and 1 ng / mL, respectively.

[0086] 3.2 Measurement results

[0087] The N-terminal and C-terminal telopeptide characteristic polypeptides in bovine type I collagen samples were detected, and the spectra were shown in Figure 2. Figure 14 、 Figure 15 The telopeptide contents were calculated according to formula (1) and formula (2). The results showed that the average contents of N-terminal and C-terminal telopeptides in bovine type I collagen samples were 16.037±0.439μg / mg and 14.087±0.125μg / mg, respectively.

[0088] The foregoing is merely a preferred embodiment of the present invention. It should be noted that, without departing from the principles of the present invention, those skilled in the art may make various modifications, combinations, alterations, or substitutions to the details and features of the present invention. Such modifications, combinations, alterations, or substitutions are considered to be within the scope of the present invention.

Claims

1. A bovine type I collagen terminal telopeptide characteristic polypeptide, characterized in that: The bovine type I collagen terminal peptide characteristic polypeptide includes an N-terminal peptide characteristic polypeptide and a C-terminal peptide characteristic polypeptide. The amino acid sequence of the N-terminal peptide characteristic polypeptide is Q*LSYGYDE, and the amino acid sequence of the C-terminal peptide is LSFLPQPPQE.

2. Use of the bovine type I collagen terminal telopeptide characteristic polypeptide according to claim 1 in detecting the N-terminal telopeptide and C-terminal telopeptide of bovine type I collagen.

3. The use according to claim 2, characterized in that The detection method is liquid chromatography-tandem mass spectrometry.

4. The use according to claim 3, characterized in that The detection method is qualitative detection, and the liquid chromatography-tandem mass spectrometry detection method comprises the following steps: 1) preparing the N-terminal telopeptide characteristic polypeptide and the C-terminal telopeptide characteristic polypeptide into solution A and solution B of certain concentrations respectively; 2) Injecting the solution A, the solution B and the sample to be tested into a liquid chromatography-mass spectrometer respectively; 3) If the retention time of the daughter ion in the sample to be tested is the same as the retention time of the daughter ion of the N-terminal telopeptide characteristic polypeptide and / or the C-terminal telopeptide characteristic polypeptide, then the N-terminal telopeptide characteristic polypeptide and / or the C-terminal telopeptide characteristic polypeptide is present in the sample to be tested.

5. The use according to claim 4, characterized in that The daughter ion of the N-terminal telopeptide characteristic polypeptide is m / z 957.38→532.03,695.19, and the daughter ion of the C-terminal telopeptide characteristic polypeptide is m / z 1155.58→470.03,695.23, which are used as detection ions to detect retention time.

6. The use according to claim 3, characterized in that The detection method is quantitative detection, and the liquid chromatography-tandem mass spectrometry detection method comprises the following steps: A. Developing a concentration standard curve: Prepare the N-terminal telopeptide characteristic polypeptide and the C-terminal telopeptide characteristic polypeptide into standard solutions of different concentrations, and perform liquid chromatography-tandem mass spectrometry detection to draw a concentration standard curve. 2 The standard curve of concentration and peak area is greater than 0.99; B. Detecting the sample by liquid chromatography-tandem mass spectrometry; C. Calculate the C-terminal telopeptide content of bovine type I collagen in the sample according to formula (1), and calculate the N-terminal telopeptide content of bovine type I collagen in the sample according to formula (2): Where: W C : C-terminal telopeptide content of bovine type I collagen in the sample to be tested, μg / mg; X C : concentration of bovine type I collagen C-terminal telopeptide characteristic polypeptide, μg / mL; V: final volume of the sample to be tested, mL; M C : Molecular weight of bovine type I collagen C-terminal telopeptide, u; M CP : Molecular weight of the characteristic polypeptide of bovine type I collagen C-terminal telopeptide, u; m: Mass of the sample to be tested, mg; n C : The number of repetitions of the C-terminal telopeptide characteristic polypeptide in bovine type I collagen; Where: W N : N-terminal telopeptide content of bovine type I collagen in the sample to be tested, μg / mg; X N : concentration of bovine type I collagen N-terminal telopeptide characteristic polypeptide, μg / mL; V: final volume of the sample to be tested, mL; M N : molecular weight of bovine type I collagen N-terminal telopeptide, u; M NP : molecular weight of bovine type I collagen N-terminal telopeptide characteristic polypeptide, u; m: mass of the sample to be tested, mg; n N The number of repeats of the N-terminal telopeptide signature polypeptide in bovine type I collagen.

7. The use according to claim 6, characterized in that The daughter ion of the N-terminal telopeptide characteristic polypeptide is m / z 957.38→532.03,695.19, and the daughter ion of the C-terminal telopeptide characteristic polypeptide is m / z 1155.58→470.03,695.23, which are used as detection ions to detect retention time.

8. The use according to any one of claims 4 to 7, characterized in that A Tris-HCl solution is used to prepare a solution of the N-terminal telopeptide characteristic polypeptide or a solution of the C-terminal telopeptide characteristic polypeptide.

9. The use according to any one of claims 4 to 7, characterized in that The mass spectrometry analysis of the N-terminal terminal peptide characteristic polypeptide and the C-terminal terminal peptide characteristic polypeptide are both electrospray ionization, positive ion mode, multiple reaction monitoring; preferably, the liquid chromatography parameters are: Zorbax C18 column; mobile phase: A is water containing 0.1% formic acid, B is an aqueous solution of 60% acetonitrile containing 0.1% formic acid (V / V); gradient elution: 0-0.5 min (5% B); 0.5-2 min (5%-45% B), 2-3 min (45%-90% B), 3-4.5 min (90%-100% B), 4.5-7 min (100% B), 7-7.1 (100%-5% B); 7.1-10 min (5% B); flow rate 0.2 ml / min; column temperature: 30°C; sample load: 5.0 μL; preferably, the mass spectrometry analysis conditions are: ion source spray voltage 3.5 kV, capillary temperature 320°C, evaporation temperature 350°C, sheath gas flow rate 19.5 mL / min, auxiliary flow rate 0.37 bar, positive ion mode scanning, SRM monitoring mode: the N-terminal terminal peptide characteristic polypeptide m / z 957.38 single charge → 532.03, the C-terminal terminal peptide characteristic polypeptide m / z 1155.58 single charge → 470.

03.

10. A method for preparing a collagenase hydrolyzate containing the bovine type I collagen terminal telopeptide characteristic polypeptide according to claim 1, characterized in that: The preparation method comprises the following steps: 1) Heat denature bovine type I collagen in a boiling water bath for 20-40 minutes to disaggregate the triple helical structure; 2) Enzymatic digestion of bovine type I collagen using Glu-C for 16-20 h at a temperature of 35-40°C; 3) analyzing the obtained enzymatic hydrolysis products using a mass spectrometer to obtain a total ion current of the bovine type I collagen enzymatic hydrolysis products; 4) importing the sequence of bovine type I collagen into BioFinder software, and aligning the obtained spectrum with the sequence of bovine type I collagen using BioFinder software; 5) The comparison results showed that the enzymatic hydrolysis product of bovine type I collagen contained an ion of m / z 1155.58 in the primary mass spectrum, with a molecular weight of 1154.60 Da, which was consistent with the theoretical molecular weight of the polypeptide LSFLPQPPQE; in the secondary mass spectrum of m / z 1155.58, its fragment ion was highly matched with the theoretical fragment ion of the polypeptide LSFLPQPPQE, indicating that the polypeptide LSFLPQPPQE only existed in the C-terminal telopeptide of bovine type I collagen; the enzymatic hydrolysis product of bovine type I collagen contained an ion of m / z 957.38 in the primary mass spectrum, with a molecular weight of 956.4, which was consistent with the theoretical molecular weight of the polypeptide Q*LSYGYDE; in the secondary mass spectrum of m / z 957.38, its fragment ion was highly matched with the theoretical fragment ion of the polypeptide Q*LSYGYDE, indicating that the polypeptide Q*LSYGYDE only existed in the N-terminal telopeptide of bovine type I collagen.

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