Protein composition analysis method for succinyl gelatin injection

Abstract

The invention relates to the technical field of protein composition analysis, in particular to a protein composition analysis method for succinyl gelatin injection, which comprises the following steps: respectively preparing a mobile phase and a plurality of reagents; the method comprises the following steps: pretreating a plurality of succinyl gelatin injection samples to obtain a plurality of peptide fragments; setting chromatographic conditions and mass spectrum conditions; separating the peptide fragment through a chromatographic instrument, and eluting according to an elution procedure; collecting data of the eluted peptide fragment by adopting a mass spectrometer to obtain a primary mass spectrum and a secondary mass spectrum; comparing the obtained mass spectrum with a database through software, and identifying the protein composition in the sample; and judging the qualification of the identification process according to the protein component consistency index, and increasing the number of samples according to the difference value between the preset consistency index and the protein component consistency index based on the unqualified condition. According to the invention, the accuracy of protein composition analysis is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein composition analysis, in particular to a protein composition analysis method for succinylated gelatin injection. Background Art

[0002] Succinylated gelatin injection, a widely used plasma substitute in clinical practice, plays a key role in expanding blood volume, maintaining blood colloidal osmotic pressure, and improving microcirculation. Its core ingredient is succinylated gelatin protein, and the characteristics of its protein composition directly determine the quality, efficacy, and safety of the injection. Succinylated gelatin injections from different batches and sources may have different protein compositions. If these differences exceed a certain range, they may affect the drug's metabolism in the body, trigger an immune response, and thus affect clinical treatment outcomes and even threaten the patient's life and health. Therefore, accurate and comprehensive analysis of the protein composition of succinylated gelatin injection is extremely important for ensuring product quality, ensuring safe and effective clinical use, and promoting the research and development and improvement of related formulations.

[0003] Chinese Patent Publication No.: CN113272649B discloses a method for determining the stoichiometry of protein components of intact unenveloped viral particles, the method comprising: performing hydrophilic interaction liquid chromatography (HILIC) on a sample containing the viral particles to separate the protein components of the viral capsid of the viral particles; determining the mass of the protein components of the viral capsid to identify the protein components separated by HILIC; and determining the relative abundance of the protein components from the viral capsid separated by HILIC, thereby determining the stoichiometry of the protein components of the viral capsid of the viral particles.

[0004] However, the existing technology has the following problems: the existing technology lacks pre-processing and determination of identification results, resulting in inaccurate protein component measurement results. Summary of the Invention

[0005] To this end, the present invention provides a method for analyzing the protein composition of succinylated gelatin injection, so as to overcome the problem in the prior art of inaccurate protein composition identification due to the lack of determination of the identification process.

[0006] To achieve the above object, the present invention provides a method for analyzing the protein composition of succinylated gelatin injection, comprising: Prepare chromatographic mobile phase and several reagents respectively; Several peptides were obtained by pre-treating several succinylated gelatin injection samples; Set up chromatographic and mass spectrometry conditions; After the peptides are separated by chromatography, they are eluted according to the elution procedure; A mass spectrometer was used to collect data of the eluted peptides to obtain primary mass spectra and secondary mass spectra; The software compares the acquired mass spectrum with the database to identify the protein composition in the sample; The eligibility of the identification process is determined according to the protein component consistency index. Based on the unqualified condition, the number of samples is increased according to the difference between the preset consistency index and the protein component consistency index.

[0007] Furthermore, the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is 0.1% formic acid-water solution, and mobile phase B is 0.1% formic acid-acetonitrile solution.

[0008] Furthermore, the reagents include NH4HCO3 solution, DTT solution, IAM solution, Rapigest SF (0.1%) solution and Trypsin solution.

[0009] Furthermore, the pretreatment process includes: pretreatment, denaturation, reduction, alkylation, termination of alkylation, enzyme digestion and quenching.

[0010] Furthermore, the chromatographic conditions include: chromatographic column: ACQUITYTM Premier peptide CSH C18, 1.7 μm, 2.1 mm×150 mm; flow rate: 0.2 mL / min; injection volume: 20 μL; autosampler temperature: 8°C; column temperature: 50°C.

[0011] Furthermore, the mass spectrometry conditions include: ion source: electrospray ion source; ionization mode: positive ion mode; sheath gas flow rate: 30 arb; auxiliary gas flow rate: 10 arb; spray voltage: 3.8 kV; capillary temperature: 300°C; auxiliary gas heater temperature: 280°C; resolution: Full MS 70,000 FWHM, ddMS2 17,500 FWHM; scan range: 200 m / z-2000 m / z.

[0012] Furthermore, the eligibility of the identification process is determined according to the protein component consistency index, wherein if the protein component consistency index is less than a preset consistency index, the identification process is determined to be unqualified, and the number of samples is increased according to the difference between the preset consistency index and the protein component consistency index; If the protein component consistency index is greater than or equal to the preset consistency index, the identification process is determined to be qualified and the sample protein composition information is recorded.

[0013] Furthermore, the number of samples is positively correlated with the consistency index difference, wherein the consistency index difference is the difference between the preset consistency index and the protein component consistency index.

[0014] Furthermore, the protein component consistency index is determined by the protein type overlap and protein content fluctuation value.

[0015] Furthermore, Proteome Discoverer software was used to search the bovine protein database to identify the protein components in the samples.

[0016] Compared with the prior art, the present invention has the beneficial effect that, through comprehensive pre-treatment, the present invention can fully destroy the structure of the sample protein, making it better adapted to subsequent chromatography and mass spectrometry analysis, thereby improving the detection rate and identification accuracy of the protein.

[0017] Furthermore, the present invention precisely sets the chromatographic conditions and mass spectrometry conditions, providing an optimal environment for the separation and detection of peptide segments, and facilitating the acquisition of high-quality mass spectrometry data.

[0018] Furthermore, the present invention uses professional software to compare the acquired mass spectrogram with a database to identify the protein components in the sample, which can improve the accuracy and specificity of protein identification and reduce false positive and false negative results.

[0019] Furthermore, the present invention constructs a protein component consistency index by introducing two core indicators: protein type overlap and protein content fluctuation value. This allows for a comprehensive and quantitative evaluation of succinylated gelatin injection from the two dimensions of protein type and content. Protein type overlap can accurately reflect the similarity of protein types in different samples, ensuring the stable presence of key protein components in the product; protein content fluctuation value can accurately measure the changes in the content of each protein between different batches, and promptly detect possible quality fluctuations. This comprehensive evaluation method overcomes the limitations of existing technologies that rely solely on qualitative analysis of characteristic peptides, providing a more scientific and rigorous basis for product quality control.

[0020] Furthermore, the present invention dynamically adjusts the number of samples based on the difference between the protein component consistency index and a preset consistency index. If the protein component consistency index is less than the preset consistency index, the identification process is deemed unqualified, and the number of samples is increased based on the difference; the number of samples is positively correlated with the difference in consistency index. This dynamic adjustment mechanism can flexibly increase the number of samples based on actual conditions, improve the stability and accuracy of the identification results, and ensure that the protein composition information ultimately obtained is reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flow chart of a method for analyzing the protein composition of succinylated gelatin injection according to an embodiment of the present invention; Figure 2 Schematic diagram of protein distribution in several samples according to the present invention; Figure 3 and Figure 4BPC diagrams of several samples of the embodiments of the present invention; Figure 5 The figure is a flow chart of determining the eligibility of an identification process according to a protein component consistency index according to an embodiment of the present invention. DETAILED DESCRIPTION

[0022] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0023] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0024] It should be pointed out that the data in this embodiment are obtained by comprehensive analysis and evaluation of the historical test data and the corresponding historical test results of the present invention in the three months before this test. It can be understood by those skilled in the art that the present invention can determine the above parameters for a single item by selecting the value with the highest proportion as the preset standard parameter based on the data distribution, using weighted summation to use the obtained value as the preset standard parameter, substituting each historical data into a specific formula and using the value obtained by the formula as the preset standard parameter or other selection methods, as long as the present invention can clearly define the different specific situations in the single determination process through the obtained values.

[0025] It should be noted that the data in this embodiment are all obtained through preliminary experimental verification using the method described in the present invention before this test. The preset values ​​can be adjusted according to specific usage conditions, as long as the method described in the present invention can clearly define the different specific situations in the single determination process through the obtained values. The preset values ​​set in this embodiment are all obtained based on preliminary experiments, including the various correction coefficients, which were also selected through experimental verification.

[0026] See also Figures 1 to 5 As shown, they are respectively a flow chart of the protein composition analysis method for succinylated gelatin injection according to an embodiment of the present invention; a schematic diagram of the distribution of protein proportions in several samples according to an embodiment of the present invention; a BPC diagram of several samples according to an embodiment of the present invention; and a flow chart of determining the eligibility of an identification process according to a protein component consistency index according to an embodiment of the present invention.

[0027] The protein composition analysis method for succinylated gelatin injection according to an embodiment of the present invention comprises: Step S1, respectively preparing a chromatographic mobile phase and several reagents; Step S2, pre-treating a plurality of succinylated gelatin injection samples to obtain a plurality of peptide segments; Step S3, setting chromatographic conditions and mass spectrometry conditions; Step S4, separating the peptides by chromatography and eluting according to the elution procedure; Step S5, using a mass spectrometer to collect data of the eluted peptides to obtain a primary mass spectrum and a secondary mass spectrum; Step S6, comparing the obtained mass spectrum with the database through software to identify the protein composition in the sample; Step S7, judging the eligibility of the identification process according to the protein component consistency index, and based on the unqualified condition, increasing the number of samples according to the difference between the preset consistency index and the protein component consistency index.

[0028] Specifically, the chromatographic mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is 0.1% formic acid-water solution, and mobile phase B is 0.1% formic acid-acetonitrile solution.

[0029] Specifically, the preparation process of mobile phase A includes: Use a measuring cylinder to measure 1000 mL of pure water into the mobile phase bottle, then use a pipette to add 1 mL of formic acid and mix well to obtain mobile phase A. The preparation process of mobile phase B includes: Use a measuring cylinder to measure 1000 mL of acetonitrile into the mobile phase bottle, then add 1 mL of formic acid using a pipette and mix well to obtain mobile phase B.

[0030] Specifically, the reagents include NH4HCO3 solution, DTT solution, IAM solution, Rapigest SF (0.1%) solution and Trypsin solution.

[0031] Specifically, the preparation process of each reagent includes: 50 mM NH4HCO3 solution: Weigh 20.12 mg of NH4HCO3 into a 15 mL centrifuge tube, add 5 mL of pure water, and mix well.

[0032] 500 mM DTT solution: Weigh 40.07 mg of DTT into a 1.5 mL centrifuge tube. Use a pipette to measure 256 μL of pure water and add it to the solution. Mix well to obtain a DTT concentration of approximately 1 M. Use a pipette to measure 100 μL of 1 M DTT and add 100 μL of pure water. Mix well to obtain 500 mM DTT solution.

[0033] 500 mM IAM solution: Weigh 92.25 mg of IAM into a 1.5 mL centrifuge tube. Use a pipette to measure 500 μL of pure water and add it to the solution. Mix thoroughly to obtain an IAM solution with a concentration of approximately 1 M. Use a pipette to measure 100 μL of 1 M IAM and add 100 μL of pure water. Mix thoroughly to obtain a 500 mM IAM solution.

[0034] Rapigest SF (0.1%) solution: Take one 1 mg vial of Rapigest SF lyophilized powder, add 1000 µL of purified water, and mix by pipetting up and down 5-10 times with a pipette.

[0035] 0.5 µg / µL Trypsin solution: Take one 25 µg vial of lyophilized Trypsin powder, add 50 µL of purified water, and mix thoroughly by pipetting up and down 5-10 times.

[0036] 10% formic acid aqueous solution: Use a pipette to measure 100 µL of formic acid, add 900 µL of pure water, and mix well.

[0037] Specifically, the pretreatment process includes: pretreatment, denaturation, reduction, alkylation, termination of alkylation, enzyme digestion and quenching.

[0038] Specifically, a preset number of succinylated gelatin injection samples are selected, and the specific process of pre-treatment for each sample includes: Pretreatment: 375 μL of succinylated gelatin injection was placed in a 1.5 mL EP tube, 625 μL of 50 mM NH4HCO3 solution was added, and the tube was heated at 80°C for 10 min. Denaturation: Take 50 µL of pretreated sample, add 50 µL of Rapigest SF surfactant (0.1%), mix, and heat at 80°C for 10 min; Reduction: Add 2 µL of 500 mM DTT solution (final concentration approximately 10 mM) to the denaturing solution and react at 60°C for 37 min. Alkylation: Add 4 µL of 500 mM IAM solution (final concentration approximately 20 mM) to the reducing solution and incubate at room temperature in the dark for 30 min. Terminate alkylation: Add 2 µL of 500 mM DTT solution to the alkylation solution and mix well. Enzyme digestion: Add 10 µL of 0.5 µg / µL Trypsin to the terminated alkylation solution and digest overnight at 37°C for 12 h. Quenching: Add 10 µL of 10% formic acid solution to the digestion solution, vortex mix for 30 s, let stand at room temperature for 30 min, and then centrifuge (14,000 rpm, 10 min, 4°C). Transfer the supernatant to an injection vial for LC-MS / MS analysis.

[0039] In the embodiment of the present invention, the preset number is set to 9, but the value is not limited thereto, and those skilled in the art may adjust the value according to actual needs.

[0040] Specifically, the chromatographic conditions include: chromatographic column: ACQUITYTM Premier peptide CSH C18, 1.7 μm, 2.1 mm×150 mm; flow rate: 0.2 mL / min; injection volume: 20 μL; autosampler temperature: 8°C; column temperature: 50°C.

[0041] Specifically, the Vanquish™ Flex UHPLC system was selected for chromatography.

[0042] Specifically, the elution procedure is shown in Table 1.

[0043] Table 1 Elution process flow chart

[0044] Specifically, the mass spectrometry conditions include: ion source: electrospray ion source; ionization mode: positive ion mode; sheath gas flow rate: 30 arb; auxiliary gas flow rate: 10 arb; spray voltage: 3.8 kV; capillary temperature: 300°C; auxiliary gas heater temperature: 280°C; resolution: Full MS 70,000 FWHM, ddMS2 17,500 FWHM; scan range: 200 m / z-2000 m / z.

[0045] Specifically, Full MS is the primary mass spectrum, and ddMS2 is the secondary mass spectrum.

[0046] Specifically, the mass spectrometer selected was a Q Exactive Plus mass spectrometer.

[0047] Specifically, the mass spectrometer parameters also include: automatic gain control target, 3e6 for the first stage; 1e5 for the second stage; maximum feed time: 100 ms for the first stage; 200 ms for the second stage.

[0048] Specifically, the eligibility of the identification process is determined according to the protein component consistency index, wherein, if the protein component consistency index is less than a preset consistency index, the identification process is determined to be unqualified, and the number of samples is increased according to the difference between the preset consistency index and the protein component consistency index; If the protein component consistency index is greater than or equal to the preset consistency index, the identification process is determined to be qualified and the sample protein composition information is recorded.

[0049] In the embodiment of the present invention, the preset consistency index is set to 0.9, but the value is not limited thereto, and those skilled in the art may adjust the value according to actual needs.

[0050] Specifically, when the protein component consistency index is lower than the preset threshold, the system automatically determines that the identification is unqualified and dynamically adjusts the sample quantity based on the difference, effectively reducing false negative / false positive results and improving the credibility of the data; the sample size is only increased when the consistency does not meet the standard, avoiding the blind expansion of the detection scale, saving time and costs, and ensuring statistical effectiveness; the protein composition information of qualified samples is systematically recorded, providing a standardized data basis for subsequent analysis and improving research repeatability.

[0051] Specifically, the number of samples is positively correlated with the consistency index difference, wherein, if the consistency index difference is less than the first preset difference, the first adjustment coefficient of 1.3 is used to increase the number of samples to the corresponding value; If the consistency index difference is greater than or equal to the first preset difference and less than the second preset difference, the second adjustment coefficient of 1.6 is used to increase the number of samples to the corresponding value; If the consistency index difference is greater than or equal to the second preset difference, the number of samples is increased to the corresponding value using a third adjustment coefficient of 2.2; The consistency index difference is the difference between the preset consistency index and the protein component consistency index.

[0052] In the embodiment of the present invention, the first preset difference is 0.12, and the second preset difference is 0.25, but the above values ​​are not limited thereto, and those skilled in the art may adjust the above values ​​according to actual needs.

[0053] Specifically, the protein component consistency index is determined by the protein type overlap and the protein content fluctuation value. The protein component consistency index = the first weight coefficient × protein type overlap + the second weight coefficient × protein content fluctuation value, wherein the first weight coefficient is 0.3, the protein type overlap = the number of common protein types between samples / the total number of protein types identified in all samples, the second weight coefficient is 0.7, and the protein content fluctuation value = the fluctuation threshold / the mean of the standard deviation of the percentage content of each protein in the common protein, and the fluctuation threshold is 0.0025.

[0054] Specifically, the present invention measures the similarity of protein types between samples by protein type overlap to ensure that key proteins are not missed; and quantifies the expression stability of common proteins by protein content fluctuation value to reduce the impact of batch effect or experimental error.

[0055] The protein composition analysis method of the embodiment of the present invention is used to detect the consistency of protein components between different batches of the same product. Using the protein component consistency index to determine the eligibility of the identification process can avoid errors in the test results caused by problems in the analysis method, such as insufficient sample quantity, and failure to detect a certain protein due to low content.

[0056] Specifically, the embodiment of the present invention uses Proteome Discoverer software to search the bovine protein database to identify the protein components in the sample.

[0057] Specifically, the search parameters of the database are shown in Table 2.

[0058] Table 2 Database search parameters item parameter Protein Library Bovine protein library (Swiss-Prots database, containing 6034 proteins) enzymes Trypsin Number of missed cuts 2 Minimum peptide length 4 Fixed modification Carbamidomethyl C Variable modifications Hydroxy / +15.995 Da (K, P)Succinyl / 100.016 (K, Peptide N-terminus) Search library first level quality deviation 10 ppm Search library secondary quality deviation 0.02 Da Specifically, the succinylated gelatin injection samples in this example were sourced from Jilin Changyuan Pharmaceutical Co., Ltd., and the batch numbers of the samples are shown in Table 3.

[0059] Table 3 Sample batch number table name batch number Succinylated gelatin injection 035D2023122111 Succinylated gelatin injection 035D2023122121 Succinylated gelatin injection 035D2023122211 Succinylated gelatin injection 035D2024011011 Succinylated gelatin injection 035D2024011111 Succinylated gelatin injection 035D2024011211 Succinylated gelatin injection 035D2024011311 Succinylated gelatin injection 035D2024011811 Succinylated gelatin injection 035D2024011911 Specifically, Figure 3 The batch numbers of the samples from top to bottom are: 035D2023122111, 035D2023122121, 035D2023122211, 035D2024011011 and 035D2024011111, Figure 4 The sample batch numbers from top to bottom are: 035D2024011211, 035D2024011311, 035D2024011811 and 035D2024011911.

[0060] In the embodiment of the present invention, the bovine protein library was searched by Proteome Discoverer software, and collagen was screened from the identification results. The identified proteins contained more than or equal to 2 characteristic peptides. A total of 11 collagen subtypes were identified, and the collagen subtypes (protein descriptions) were P02453 (Collagen alpha-1(I) chain), P02465 (Collagen alpha-2(I) chain), P02459 (Collagen alpha-1(II) chain), P04258 (Collagen alpha-1(III) chain), Q7SIB2 (Collagen alpha-1(IV) chain), Q32S24 (Collagen alpha-2(XI) chain), C0HLN2 (Collagen alpha-2(IX) chain), Q28083 (Collagen alpha-1(XI) chain), P23206 (Collagen alpha-1(X) chain), A6QPB3 (Collagen alpha-2(IX) chain). alpha-1(XVII) chain), Q29442 (Collagen alpha-4(IV) chain), the protein identification ratios in each sample are shown in Table 4 and Figure 2 shown.

[0061] Specifically, sequence coverage is calculated by dividing the number of detected amino acid residues by the total number of amino acid residues (the theoretical number of amino acids in a protein), and then multiplying by 100%. Sequence coverage = ND / NTotal, where N represents the number of amino acid residues, ND represents the number of detected amino acid residues, and NTotal represents the theoretical number of amino acids.

[0062] Table 4 Protein identification percentage in each sample

[0063] Table 5 Standard deviation of the percentage of each protein in the common protein

[0064] In the present embodiment, for all the above samples, the number of shared protein species among the samples was 11, and the total number of protein species identified in all samples was 11, so the protein species overlap was 1; the mean of the standard deviation of the percentage content of each protein in the shared protein was 0.0026, so the protein content fluctuation value was 0.96. In summary, the protein component consistency index among the samples in this embodiment was 0.97, and the identification process was judged to be qualified, proving that the sample capacity of this embodiment met the standards and the protein components among the samples were highly consistent.

[0065] Specifically, from Table 4 and Figure 2 As can be seen in the results, the protein composition of each batch of succinylated gelatin injection showed that the relative content of each component fluctuated within a range of less than ±3%, demonstrating the consistency of protein composition across batches. Furthermore, the low volatility of the test results demonstrates the stability of the protein composition analysis method for succinylated gelatin injection described in this embodiment of the present invention. If the analysis method were subject to systematic or random errors, significant differences would be observed in the test results of different batches of samples. However, the minimal fluctuation of ±3% observed demonstrates the excellent repeatability and reproducibility of the method.

[0066] Specifically, the embodiments of the present invention achieve high-precision and high-sensitivity detection of the complex protein composition of succinylated gelatin injection by optimizing the separation conditions and mass spectrometry detection parameters of the Vanquish™ Flex UHPLC system and combining it with an innovative protein component consistency index evaluation system.

[0067] Thus far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art may make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will fall within the scope of protection of the present invention.

[0068] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for analyzing the protein composition of succinylated gelatin injection, characterized in that: include: Prepare chromatographic mobile phase and several reagents respectively; Several succinylated gelatin injection samples were pre-treated to obtain several peptide fragments; Set up chromatographic and mass spectrometry conditions; After the peptides are separated by chromatography, they are eluted according to the elution procedure; A mass spectrometer was used to collect data of the eluted peptides to obtain primary mass spectra and secondary mass spectra; The obtained mass spectra were compared with the bovine protein database to identify the protein composition of the sample; The eligibility of the identification process is determined based on the protein component consistency index determined by the consistency of the protein species and the volatility of the protein content. Based on the unqualified condition, the number of samples is increased according to the difference between the preset consistency index and the protein component consistency index, and the mass spectrum is re-obtained to redetermine the protein composition; The protein component consistency index is jointly determined by the protein type overlap and the protein content fluctuation value. The protein component consistency index = the first weight coefficient × protein type overlap + the second weight coefficient × protein content fluctuation value, wherein the protein type overlap = the number of common protein types between samples / the total number of protein types identified in all samples, and the protein content fluctuation value = the fluctuation threshold / the mean of the standard deviation of the percentage content of each protein in the common protein.

2. The protein composition analysis method for succinylated gelatin injection according to claim 1, characterized in that: The chromatographic mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is 0.1% formic acid-water solution, and mobile phase B is 0.1% formic acid-acetonitrile solution.

3. The protein composition analysis method for succinylated gelatin injection according to claim 2, characterized in that: Several reagents include NH4HCO3 solution, DTT solution, IAM solution, Rapigest SF (0.1%) solution and Trypsin solution.

4. The protein composition analysis method for succinylated gelatin injection according to claim 3, characterized in that: The pretreatment process includes: pretreatment, denaturation, reduction, alkylation, termination of alkylation, enzyme digestion and quenching.

5. The protein composition analysis method for succinylated gelatin injection according to claim 4, characterized in that: The chromatographic conditions include: chromatographic column: ACQUITYTM Premier peptide CSH C18, 1.7 μm, 2.1 mm×150 mm; flow rate: 0.2 mL / min; injection volume: 20 μL; autosampler temperature: 8°C; column temperature: 50°C.

6. The protein composition analysis method for succinylated gelatin injection according to claim 5, characterized in that: The mass spectrometry conditions include: ion source: electrospray ion source; ionization mode: positive ion mode; sheath gas flow rate: 30 arb; auxiliary gas flow rate: 10 arb; spray voltage: 3.8 kV; capillary temperature: 300°C; auxiliary gas heater temperature: 280°C; resolution: Full MS 70,000 FWHM, ddMS2 17,500 FWHM; scan range: 200 m / z-2000 m / z.

7. The protein composition analysis method for succinylated gelatin injection according to claim 6, characterized in that: The eligibility of the identification process is determined based on the protein component consistency index, where: If the protein component consistency index is less than the preset consistency index, the identification process is determined to be unqualified, and the number of samples is increased according to the difference between the preset consistency index and the protein component consistency index; If the protein component consistency index is greater than or equal to the preset consistency index, the identification process is determined to be qualified and the sample protein composition information is recorded.

8. The protein composition analysis method for succinylated gelatin injection according to claim 7, characterized in that: The sample quantity is positively correlated with the consistency index difference, wherein the consistency index difference is the difference between the preset consistency index and the protein component consistency index.

9. The protein composition analysis method for succinylated gelatin injection according to claim 8, characterized in that: The protein component consistency index is determined by the protein type overlap and protein content fluctuation value.

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