Collagen type and content detection method and application thereof
By treating fish glue with alkali, lysing with lysis buffer, and detecting with nano-level liquid chromatography, the problem of difficulty in quantifying collagen subtypes was solved, and the stability and accuracy of collagen data were achieved.
Patent Information
- Application Number
- CN202511042840.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-10-31
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing technologies cannot effectively quantify collagen subtypes, and variations in sample pretreatment in liquid chromatography-mass spectrometry (LC-MS) lead to unstable data. Enzyme-linked immunosorbent assay (ELISA) is complex to operate and has large errors, while gel electrophoresis has insufficient separation.
After alkaline soaking, fat was removed with n-butanol, collagen was lysed with lysis buffer, and after column separation and desalting, it was detected by nano-level liquid chromatography and Q-Exactive high-resolution mass spectrometry, combined with Proteome Discoverer 2.5 software analysis.
By optimizing pretreatment and mass spectrometry detection, we ensured stable collagen data, clarified the content and types of all collagen subtypes in fish glue, and avoided misjudgment and salt ion interference in traditional methods.
Smart Images
Figure CN120870384A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of collagen detection technology, and in particular to a method and application for detecting collagen type and content. Background Technology
[0002] Collagen has nearly 30 subtypes, each with different functions. While the hydroxyproline method can detect collagen content, it cannot quantify subtypes. Currently, methods for quantifying collagen subtypes include gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-mass spectrometry (LC-MS). Gel electrophoresis can only distinguish collagen subtypes based on molecular weight, lacking precise separation for subtypes with similar molecular weights. LC-MS results in unstable subtype content data due to differences between sample and pretreatment; for example, conventional pretreatment only uses urea denaturation, failing to consider the influence of the redox environment on collagen structure. ELISA requires the development of specific antibodies for different subtypes from different organisms, making it complex and prone to significant errors. However, in terms of error severity, LC-MS < ELISA < gel electrophoresis.
[0003] Fish maw (dried fish bladder) is rich in collagen, with a collagen content that can reach up to 95%. This collagen is significantly different from ordinary protein samples, and fish maw contains different subtypes of collagen. Therefore, clarifying the types and contents of collagen in fish maw is beneficial for better utilization of fish maw collagen. Summary of the Invention
[0004] This application provides a method and application for detecting collagen type and content to solve the problems existing in related technologies. The technical solution is as follows:
[0005] In a first aspect, embodiments of this application provide a method for detecting collagen type and content, comprising the following steps:
[0006] After soaking in alkali, fish maw is treated with n-butanol to remove fat; the collagen is then lysed and dissolved using a lysis buffer, and the supernatant obtained after centrifugation is the collagen solution.
[0007] The collagen solution was hydrolyzed with protease; after hydrolysis, the eluent was obtained by column separation and desalting; the eluent was separated by nano-level liquid chromatography and then detected by Q-Exactive high-resolution mass spectrometry.
[0008] The raw mass spectrometry test files were searched in the database using Proteome Discoverer 2.5 software to obtain collagen identification results.
[0009] In one embodiment, the fish glue is first cut into small pieces before being soaked in alkali, then soaked in water at 2-8°C for 2-3 days, and then ground into powder in a liquid nitrogen environment.
[0010] In one embodiment, the alkali used for alkali soaking is a sodium hydroxide solution with a concentration of 0.05-0.2M; the mass-to-volume ratio of fish glue powder to sodium hydroxide aqueous solution is 1 mg:(30-80) mL, and the alkali soaking time is 12-48 h.
[0011] In one embodiment, after the alkaline soaking process, the fish glue is soaked in a n-butanol solution, with the mass-to-volume ratio of fish glue powder to n-butanol being 1 mg:(30-80) mL, and the soaking time being 48-72 h.
[0012] In one embodiment, the lysis buffer comprises the following components: 6-10M urea, 100Mm Tris-HCl, 6-14Mm DTT, 0.5-3Mm PMSF, pH 8-9; wherein DTT and PMSF are freshly prepared and used immediately. Urea acts as a protein denaturant, and DTT and PMSF protect collagen integrity.
[0013] In one embodiment, the mass-to-volume ratio of the fish glue powder to the lysis solution is 0.1-0.5 g / mL;
[0014] In one embodiment, the centrifugation conditions are 12000-15000g, 2-5℃, centrifugation for 20-40min, and the supernatant is the collagen sample solution.
[0015] In one embodiment, the collagen solution is prepared before enzymatic hydrolysis with protease;
[0016] The preparation process of the enzymatic hydrolysate is as follows: DTT solution is added to the collagen sample solution, and the reaction is carried out at 35-40℃ for 0.5-2 hours; then IAM is added, and the reaction is carried out in the dark for 15-90 minutes; DTT solution is added to quench the IAM; the volume is adjusted with NH4HCO3 solution to make the final urea concentration less than 1 mol / L; the enzymatic hydrolysate is obtained. 1M DTT solution is added to bring the DTT concentration to 10 mM, and IAM is quenched.
[0017] In one embodiment, the enzymatic hydrolysis uses trypsin and / or pepsin; the hydrolysis conditions are: enzyme to protein mass ratio of 1:(30-50), hydrolyzed in a water bath at 37°C for 16-24 hours.
[0018] In one embodiment, the separation column used in the column separation and desalting process is a Waters SEP-PAK C18 solid phase extraction column; the Waters SEP-PAK C18 solid phase extraction column is pretreated with a mixed solution of methanol, formic acid and acetonitrile before use;
[0019] The column separation and desalting process is as follows: after enzymatic digestion and sample loading, the separation column is centrifuged; then 0.1% formic acid is added for redissolution; the solid phase extraction column is then eluted with 70% acetonitrile solution containing 0.1% formic acid, and the eluent is the solution of desalted peptides; after concentration, 0.1% formic acid is dissolved, centrifuged, and the supernatant is the sample to be tested.
[0020] In one embodiment, the pretreatment process is as follows: the solid-phase extraction column is centrifuged with a methanol solution, then a 70% acetonitrile solution containing 0.1% formic acid is added and centrifuged again; finally, 0.1% formic acid is added and centrifuged again to complete the pretreatment.
[0021] During the column separation and desalting process, the enzymatically digested sample is loaded and centrifuged; 0.1% formic acid is added and centrifuged again; the solid-phase extraction column is centrifuged with a 70% acetonitrile solution containing 0.1% formic acid; the eluent is the desalted peptide fragment.
[0022] The eluent was dissolved in 0.1% formic acid and centrifuged at 8000-12000g for 5-20 minutes.
[0023] In one embodiment, the chromatographic conditions for nano-level liquid chromatography separation are as follows: solution A is a 0.1% formic acid aqueous solution, and solution B is an 80% acetonitrile aqueous solution containing 0.1% formic acid;
[0024] The liquid chromatography column was 50μm*150mm, Acclaim PepMap™ RSLC, thermo scientific Technology Inc.
[0025] The sample was equilibrated with 92% solution A, and then separated by liquid chromatography with an injection volume of 1 μL.
[0026] The liquid phase gradient was set as follows: 0 min - 98 min, linear gradient of liquid B from 8% to 28%; 98 min - 113 min, linear gradient of liquid B from 28% to 37%; 113 min - 117 min, linear gradient of liquid B from 37% to 100%; 117 min - 120 min, liquid B was maintained at 100%.
[0027] In one implementation, the detection conditions for the Q-Exactive high-resolution mass spectrometer are as follows:
[0028] Mass spectrometry analysis was performed using a Thermo QE HF mass spectrometer: analysis time was 120 min; detection mode was positive ion.
[0029] The mass-charge ratio of peptides and peptide fragments was collected using the following method: 20 fragment spectra were collected after each full scan; the scan range was 400-1800, the primary resolution was 60000, the secondary resolution was 15000, and the collision energy was CE 28eV.
[0030] In one implementation, the relevant database is searched, and the parameters and descriptions used for the database search are as follows: the maximum allowed number of missed cleavage sites is 2; the fixed modification type is Carbamidomethyl modification; the variable modification types are Oxidation, Acetyl / +42.011Da, Met-loss / -131.040Da, and Met-loss+Acetyl / -89.030Da; the database name used for the database search is uniprotkb_taxonomy_id_; the primary ion mass tolerance is 10ppm; the secondary ion mass tolerance is 0.02Da; and the screening criterion for reliable proteins is ≤0.01.
[0031] Secondly, the embodiments of this application provide the application of the above-described method for detecting collagen type and content in fish collagen detection.
[0032] The advantages or beneficial effects of the above technical solutions include at least the following:
[0033] This application presents a method for detecting collagen types and their content. It optimizes the pretreatment process for fish glue with high collagen content, employing a synergistic use of collagen denaturants and protectants to maximize the preservation of the natural conformation of collagen and avoid misidentification of subtypes due to incomplete oxidation or enzymatic hydrolysis in traditional methods. Desalting is performed after enzymatic hydrolysis, resolving the problem of residual salt ions interfering with mass spectrometry detection in traditional methods, thus ensuring stable data for subsequent liquid chromatography-mass spectrometry (LC-MS). The detection method provided by this application yields comprehensive data, clearly identifying all collagen subtypes in fish glue.
[0034] The above overview is for illustrative purposes only and is not intended to be limiting in any way. Further aspects, embodiments, and features of this application will become readily apparent from the following detailed description, in addition to the illustrative aspects, embodiments, and features described above. Attached Figure Description
[0035] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.
[0036] Figure 1 The total ion chromatogram for Example 1;
[0037] Figure 2 The total ion chromatogram for Example 2;
[0038] Figure 3 The total ion chromatogram for Example 3;
[0039] Figure 4 The total ion chromatogram for Example 4;
[0040] Figure 5 This is the total ion flow chromatogram for Example 5. Detailed Implementation
[0041] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the description is considered to be exemplary in nature and not restrictive.
[0042] Some of the reagents used in the examples and their suppliers:
[0043] Trypsin (Fisher Scientific); Acetonitrile (Fisher Chemical); Methanol (Sigma); Formic acid (Macklin); Iodoacetamide (IAM) (Sigma); Dithiothreitol (DTT) (Sigma); Urea (Aladdin); Tris Solarbio; Protease inhibitor (PMSF) (Calbiochem); Triethylammonium bicarbonate (TEAB) (Sigma); Ultrapure water (H2O) (Fisher Chemical); Ammonium bicarbonate (NH4HCO3) (Macklin); Potassium chloride (KCl) (China National Pharmaceutical Group).
[0044] Instrument Model:
[0045] EASY-nLC 1200 Ultra-High Performance Liquid Chromatography System (Thermo Fisher Scientific)
[0046] High-resolution mass spectrometry Q-Exactive HF (Thermo Fisher Scientific).
[0047] Example 1
[0048] 1. Grinding and impurity removal: Take 2g of fish glue sample, cut it into small pieces, add water to submerge the sample, and refrigerate it in a refrigerator (2-8℃) for 2-3 days; take 300mg of tissue sample and grind it into powder in liquid nitrogen environment, and transfer it to a centrifuge tube; take 0.1M NaOH at a ratio of 1:50w / v and soak for 24h to remove impurities, proteins and pigments, change the solution every 12h, wash with water until neutral, and then take n-butanol at a ratio of 1:50w / v and soak for 48h to remove fat, change the solution every 24h;
[0049] 2. Collagen dissolution: After lysis, add 1 ml of lysis buffer and mix well (8 M urea, 100 Mm Tris-HCl, 10 Mm DTT, 1.5 Mm PMSF, pH 8.5, where DTT and PMSF are freshly prepared and used immediately) and then homogenize thoroughly using a homogenizer.
[0050] 3.14000g, 4℃, centrifuged for 30min, the supernatant is collected as protein sample solution, and the protein concentration is determined using BCA; this facilitates the next step of quantitative protein collection.
[0051] 4. Enzymatic digestion of sample solution:
[0052] Take 150 μg of total protein sample and place it in a centrifuge tube. Add 1 mol / L DTT solution to a final concentration of approximately 10 mmol / L and react in a 37°C water bath for 1 h. Then add an appropriate amount of 1 mol / L IAM to a final concentration of 50 mmol / L and react at room temperature in the dark for 45 min. Add 1 M DTT solution to bring the concentration to 10 mM to quench the IAM. Adjust the volume with 50 mmol / L NH4HCO3 solution to a final urea concentration of less than 1 mol / L. Add trypsin at a ratio of m(enzyme):m(protein) = 1:45. Add pepsin at a ratio of 1:50 and incubate the mixture in a 37°C water bath for 20 h.
[0053] 5. Desalting of enzymatic hydrolysis products:
[0054] Load a Waters SEP-PAK C18 solid-phase extraction column into a 15 mL centrifuge tube, add 1 mL of methanol and centrifuge at 150 rpm for 1 min, then add 0.5 mL of 70% acetonitrile (containing 0.1% formic acid, volume fraction, the same below), and centrifuge at 150 rpm for 1 min; add 0.5 mL of 0.1% formic acid, and centrifuge at 150 rpm for 1 min; load the enzymatically digested sample and centrifuge at 150 rpm for 3 min; add 0.5 mL of 0.1% formic acid, and centrifuge at 150 rpm for 3 min; remove the solid-phase extraction column, load it into a new 15 mL centrifuge tube, add 0.5 mL of 70% acetonitrile (containing 0.1% formic acid), and centrifuge at 150 rpm for 3 min, repeating once; the eluent is the desalted peptide fragment, which is concentrated by centrifugation. Dissolve in 100 μL of 0.1% formic acid, centrifuge at 10000g for 10 min, take the supernatant for nano-level liquid chromatography separation and then detect with Q-Exactive high-resolution mass spectrometer;
[0055] (2) Chromatography-mass spectrometry analysis:
[0056] ①Chromatographic separation: Solution A used in the liquid chromatography was a 0.1% formic acid aqueous solution, and Solution B was a 0.1% formic acid-acetonitrile aqueous solution (acetonitrile content 80%). The liquid chromatography column was 50μm*150mm, Acclaim PepMap™ RSLC, Thermo Scientific Technology Inc.; equilibration was performed with 92% solution A, and the injection volume was μL for separation via the chromatographic column.
[0057] The relevant liquid phase gradient settings are as follows: 0 min - 98 min, linear gradient of liquid B from 8% to 28%; 98 min - 113 min, linear gradient of liquid B from 28% to 37%; 113 min - 117 min, linear gradient of liquid B from 37% to 100%; 117 min - 120 min, liquid B is maintained at 100%.
[0058] ②Mass spectrometry identification: After separation by nano-level liquid chromatography, the enzymatic hydrolysis products were analyzed by mass spectrometry using a Thermo QE HF mass spectrometer (ThermoFisher). The analysis time was 120 min; the detection mode was positive ion; the mass-charge ratio of the peptide and peptide fragments was collected according to the following method: 20 fragment spectra were collected after each full scan (MS2 scan); the scan range was 400-1800, the primary resolution was 60000, the secondary resolution was 15000, and the collision energy was CE 28 eV.
[0059] (3) Data Analysis: The raw mass spectrometry test file was retrieved from the corresponding database (uniprotkb_taxonomy_id_310915(Pangasianodonhypophthalmus)) using Proteome Discoverer 2.5 software to obtain the collagen identification results. The total ion chromatogram is shown below. Figure 1 As shown in Table 2, the types and contents of collagen are as follows.
[0060] Table 1
[0061]
[0062]
[0063] Example 2
[0064] The difference between Example 2 and Example 1 is that the lysis buffer was 8M urea, 100Mm Tris-HCl, 8Mm DTT, 1.5Mm PMSF, pH 8.5; the enzymatic hydrolysis was performed using trypsin at a ratio of m(enzyme):m(protein) = 1:45, and the hydrolysis time was 16 hours; other conditions and methods were the same as in Example 1. The total ion chromatogram is shown below. Figure 2As shown in Table 2, the types and contents of collagen are as follows.
[0065] Example 3
[0066] The difference between Example 3 and Example 1 is that the lysis buffer was 8M urea, 100Mm Tris-HCl, 6Mm DTT, 0.5Mm PMSF, pH 8.5; the enzymatic hydrolysis time was 16h; other conditions and methods were the same as in Example 1. The total ion chromatogram is shown below. Figure 3 As shown in Table 2, the types and contents of collagen are as follows.
[0067] Example 4
[0068] The difference between Example 3 and Example 1 is that the lysis buffer is 8M urea, 100Mm Tris-HCl, 8Mm DTT, 1.5Mm PMSF, pH 8.5; the enzymatic hydrolysis uses trypsin at a ratio of m(enzyme):m(protein) = 1:45, followed by pepsin at a ratio of 1:40. Other conditions and methods are the same as in Example 1. The total ion chromatogram is shown below. Figure 4 As shown in Table 2, the types and contents of collagen are as follows.
[0069] Example 5
[0070] The difference between Example 5 and Example 1 is that the lysis buffer was 8M urea, 100Mm Tris-HCl, 6Mm DTT, 1.5Mm PMSF, pH 8.5; the enzymatic hydrolysis was performed using trypsin at a ratio of m(enzyme):m(protein) = 1:45, and the hydrolysis time was 24 hours; other conditions and methods were the same as in Example 1. The total ion chromatogram is shown below. Figure 5 As shown in Table 2, the types and contents of collagen are as follows.
[0071] Table 2
[0072]
[0073] Comparative Example 1
[0074] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not include the desalting step of the enzymatic hydrolysis product, while the other conditions and methods are the same as in Example 1.
[0075] When testing the collagen prepared in Comparative Example 1, without desalting, the salt and macromolecules could easily damage the instrument, causing column blockage and making the experiment impossible, thus requiring the test to be stopped.
[0076] Comparative Example 2
[0077] The difference between Comparative Example 2 and Example 1 is that the collagen dissolution step is not included. After protein extraction, enzymatic hydrolysis is performed directly. Other conditions and methods are the same as in Example 1.
[0078] Without collagen dissolution, collagen cannot be effectively released, resulting in serious missed detections; collagen cross-linking hinders enzymatic hydrolysis, leading to extremely low peptide recovery rates; non-specific adsorption loss results in failed collagen enrichment; generally, only common type I collagen can be detected, and the number of other non-collagen proteins is also small.
[0079] The lysis buffer combination in this application transforms collagen from an insoluble fibrous state into a soluble, linear, and complete polypeptide chain through a triple mechanism of denaturation, reduction, and degradation inhibition, laying the foundation for subsequent extraction, enzymatic digestion, mass spectrometry, and other analyses.
[0080] Example 6
[0081] The sample prepared in Example 1 was tested three times using the method described in Example 1. The results are shown in Table 3.
[0082] Table 2
[0083]
[0084] The results in Table 2 show that the deviation was small in multiple tests, indicating that the test results of the processed samples and the test methods in this application are stable.
[0085] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.
[0086] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.
[0087] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.
Claims
1. A method for detecting collagen type and content, characterized in that, Includes the following steps: Fish maw is soaked in alkali and then the fat is removed with n-butanol; The collagen is lysed and dissolved using a lysis buffer, and the supernatant obtained after centrifugation is the collagen solution. The collagen solution was hydrolyzed with protease; after hydrolysis, the eluent was obtained by column separation and desalting; the eluent was separated by nano-level liquid chromatography and then detected by Q-Exactive high-resolution mass spectrometry. The raw mass spectrometry test files were searched in the database using Proteome Discoverer 2.5 software to obtain collagen identification results.
2. The method for detecting collagen type and content according to claim 1, characterized in that, Before soaking in alkali, fish maw is first cut into small pieces, soaked in water at 2-8℃ for 2-3 days, and then ground into powder in a liquid nitrogen environment. The alkali used for soaking is a sodium hydroxide solution with a concentration of 0.05-0.2M; the mass-to-volume ratio of fish glue powder to sodium hydroxide aqueous solution is 1mg:(30-80)mL, and the soaking time is 12-48h; After the alkaline soaking is completed, the fish glue is soaked in a n-butanol solution with a mass-to-volume ratio of fish glue powder to n-butanol of 1 mg:(30-80) mL for 48-72 h.
3. The method for detecting collagen type and content according to claim 1, characterized in that, The lysis buffer consists of the following components: 6-10M urea, 100Mm Tris-HCl, 6-14Mm DTT, 0.5-3Mm PMSF, pH 8-9; DTT and PMSF should be prepared and used immediately. The mass-to-volume ratio of the fish glue powder to the lysis solution is 0.1-0.5 g / mL; The centrifugation conditions are 12000-15000g, 2-5℃, centrifugation for 20-40 minutes, and the supernatant is the collagen sample solution.
4. The method for detecting collagen type and content according to claim 1, characterized in that, Before using protease to hydrolyze collagen solution, prepare the hydrolysis solution first; The preparation process of the enzymatic hydrolysate is as follows: DTT solution is added to the collagen sample solution and reacted at 35-40℃ for 0.5-2 hours; then IAM is added and reacted in the dark for 15-90 minutes; DTT solution is added to quench the IAM; the volume is adjusted with NH4HCO3 solution to make the final urea concentration less than 1 mol / L; the enzymatic hydrolysate is obtained. The enzymatic hydrolysis uses trypsin and / or pepsin; the enzymatic hydrolysis conditions are: enzyme to protein mass ratio of 1:(30-50), and enzymatic hydrolysis at 37℃ for 16-24 hours.
5. The method for detecting collagen type and content according to claim 1, characterized in that, During the column separation and desalting process, the separation column used is a Waters SEP-PAK C18 solid phase extraction column; the Waters SEP-PAK C18 solid phase extraction column is pretreated with a mixed solution of methanol, formic acid and acetonitrile before use; The column separation and desalting process is as follows: after enzymatic digestion and sample loading, the separation column is centrifuged; then 0.1% formic acid is added for redissolution; the solid phase extraction column is then eluted with 70% acetonitrile solution containing 0.1% formic acid, and the eluent is the solution of desalted peptides; After concentration, the solution is dissolved in 0.1% formic acid, centrifuged, and the supernatant is the sample to be tested.
6. The method for detecting collagen type and content according to claim 5, characterized in that, The pretreatment process is as follows: the solid-phase extraction column is centrifuged with methanol solution, then 70% acetonitrile solution containing 0.1% formic acid is added and centrifuged again; finally, 0.1% formic acid is added and centrifuged again to complete the pretreatment. During the column separation and desalting process, the enzymatically digested sample is loaded and centrifuged; 0.1% formic acid is added and centrifuged again; the solid-phase extraction column is centrifuged with a 70% acetonitrile solution containing 0.1% formic acid; the eluent is the desalted peptide fragment. The eluent was dissolved in 0.1% formic acid and centrifuged at 8000-12000g for 5-20 minutes.
7. The method for detecting collagen type and content according to claim 1, characterized in that, The chromatographic conditions for nano-level liquid chromatography separation were as follows: Solution A was a 0.1% formic acid aqueous solution, and Solution B was an 80% acetonitrile aqueous solution containing 0.1% formic acid. The liquid chromatography column was 50μm*150mm, Acclaim PepMap™ RSLC, Thermo Scientific Technology Inc. The sample was equilibrated with 92% solution A, and then separated by liquid chromatography with an injection volume of 1 μL. The liquid phase gradient was set as follows: 0 min - 98 min, linear gradient of liquid B from 8% to 28%; 98 min - 113 min, linear gradient of liquid B from 28% to 37%; 113 min - 117 min, linear gradient of liquid B from 37% to 100%; 117 min - 120 min, liquid B was maintained at 100%.
8. The method for detecting collagen type and content according to claim 1, characterized in that, The detection conditions for the Q-Exactive high-resolution mass spectrometer are as follows: Mass spectrometry analysis was performed using a Thermo QE HF mass spectrometer: analysis time was 120 min; detection mode was positive ion. The mass-charge ratio of peptides and peptide fragments was collected using the following method: 20 fragment spectra were collected after each full scan; the scan range was 400-1800, the primary resolution was 60000, the secondary resolution was 15000, and the collision energy was CE 28eV.
9. The method for detecting collagen type and content according to claim 1, characterized in that, The relevant parameters and specifications used for the database search are as follows: the maximum allowed number of missed cleavage sites is 2; the fixed modification type is Carbamidomethyl modification; the variable modification types are Oxidation, Acetyl / +42.011Da, Met-loss / -131.040Da, and Met-loss+Acetyl / -89.030Da; the database name used for the search is uniprotkb_taxonomy_id_; the primary ion mass tolerance is 10ppm; the secondary ion mass tolerance is 0.02Da; and the screening criterion for reliable proteins is ≤0.
01.
10. The application of the method for detecting collagen type and content according to any one of claims 1-9 in the detection of fish collagen.