A method for analyzing protein composition of succinylated gelatin injection

Abstract

This invention relates to the field of protein composition analysis technology, and particularly to a method for protein composition analysis of succinyl gelatin injection solution, comprising: preparing a mobile phase and several reagents; pretreating several succinyl gelatin injection solution samples to obtain several peptide fragments; setting chromatographic and mass spectrometric conditions; separating the peptide fragments by chromatography and eluting them according to an elution program; acquiring data of the eluted peptide fragments using a mass spectrometer to obtain primary and secondary mass spectra; comparing the acquired mass spectra with a database using software to identify the protein composition in the samples; determining the pass / fail status of the identification process based on a protein component consistency index, and, based on non-compliance conditions, increasing the number of samples according to the difference between the preset consistency index and the protein component consistency index. This invention improves the accuracy of protein composition analysis.

Description

Technical Field

[0001] This invention relates to the field of protein composition analysis technology, and in particular to a method for protein composition analysis of succinyl gelatin injection. Background Technology

[0002] Succinylated gelatin injection, a widely used plasma substitute in clinical practice, plays a crucial role in expanding blood volume, maintaining blood colloid osmotic pressure, and improving microcirculation. Its core component is succinylated gelatin protein, and the characteristics of its protein composition directly determine the quality, efficacy, and safety of the injection. The protein composition of succinylated gelatin injections from different batches and sources may vary. If these differences exceed a certain range, they may affect the drug's metabolic process in the body, trigger immune responses, and thus impact clinical treatment efficacy, even threatening patient health. Therefore, accurate and comprehensive analysis of the protein composition of succinylated gelatin injection is of paramount importance for ensuring product quality, guaranteeing safe and effective clinical use, and promoting the research and development of related formulations.

[0003] Chinese Patent Publication No. CN113272649B discloses a method for determining the stoichiometry of protein components in an intact, unenveloped viral particle. The method includes: performing hydrophilic interaction liquid chromatography (HILIC) on a sample containing the viral particle to separate the protein components of the viral capsid of the viral particle; 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 particle.

[0004] However, the existing technology has the following problems: the lack of pretreatment and judgment of identification results leads to inaccurate protein component measurement results. Summary of the Invention

[0005] Therefore, the present invention provides a protein composition analysis method for succinyl gelatin injection, which overcomes the problem of inaccurate protein composition identification caused by the lack of determination of the identification process in the prior art.

[0006] To achieve the above objectives, the present invention provides a method for protein composition analysis of succinyl gelatin injection, comprising:

[0007] Prepare the chromatographic mobile phase and several reagents respectively;

[0008] Several peptide fragments were obtained by pretreatment of several succinyl gelatin injection samples.

[0009] Set chromatographic and mass spectrometric conditions;

[0010] After separating the peptides by chromatography, elution was performed according to the elution program;

[0011] Mass spectrometry was used to acquire data of the eluted peptides to obtain primary and secondary mass spectra.

[0012] The obtained mass spectra are compared with the database using software to identify the protein composition in the sample.

[0013] The pass / failure of the identification process is determined based on the protein component consistency index. If the process fails, the number of samples is increased based on the difference between the preset consistency index and the protein component consistency index.

[0014] Furthermore, the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.1% formic acid-water solution and mobile phase B is a 0.1% formic acid-acetonitrile solution.

[0015] Furthermore, several reagents include NH4HCO3 solution, DTT solution, IAM solution, Rapidest SF (0.1%) solution and Trypsin solution.

[0016] Furthermore, the pretreatment process includes: pretreatment, denaturation, reduction, alkylation, termination of alkylation, enzyme digestion and quenching.

[0017] Furthermore, the chromatographic conditions included: column: ACQUITY™ 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.

[0018] Furthermore, the mass spectrometry conditions included: ion source: electrospray ionization 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.

[0019] Furthermore, the qualification of the identification process is determined based on the protein component 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 between the preset consistency index and the protein component consistency index.

[0020] If the protein component consistency index is greater than or equal to the preset consistency index, the identification process is deemed qualified, and the sample protein composition information is recorded.

[0021] 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.

[0022] Furthermore, the protein component consistency index is determined by both the overlap of protein types and the fluctuation value of protein content.

[0023] Furthermore, the Proteome Discoverer software was used to search the bovine protein database to identify the protein components in the sample.

[0024] Compared with the prior art, the beneficial effect of the present invention is that the present invention can fully destroy the structure of sample proteins through comprehensive pretreatment, making them better suited for subsequent chromatographic and mass spectrometric analysis, thereby improving the detection rate and identification accuracy of proteins.

[0025] Furthermore, the present invention precisely sets the chromatographic and mass spectrometry conditions, providing an optimal environment for peptide separation and detection, which helps to obtain high-quality mass spectrometry data.

[0026] Furthermore, this invention uses specialized software to compare the acquired mass spectra 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.

[0027] Furthermore, this invention constructs a protein component consistency index by introducing two core indicators: protein type overlap and protein content fluctuation. This allows for a comprehensive and quantitative evaluation of succinyl gelatin injection from both protein type and content dimensions. Protein type overlap accurately reflects the similarity of protein types in different samples, ensuring the stable presence of key protein components in the product. Protein content fluctuation accurately measures the changes in the content of each protein between different batches, promptly identifying potential 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.

[0028] Furthermore, this 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 according to the difference; the number of samples is positively correlated with the difference in the consistency index. This dynamic adjustment mechanism can flexibly increase the number of samples according to the actual situation, improving the stability and accuracy of the identification results and ensuring the reliability of the final obtained protein composition information. Attached Figure Description

[0029] Figure 1 This is a flowchart of a protein composition analysis method for succinyl gelatin injection according to an embodiment of the present invention;

[0030] Figure 2 This is a schematic diagram showing the protein percentage distribution in several samples according to embodiments of the present invention;

[0031] Figure 3 and Figure 4 These are BPC diagrams of several samples from embodiments of the present invention;

[0032] Figure 5 This is a flowchart illustrating the process of determining the pass / fail status of the identification process based on the protein component consistency index, as described in an embodiment of the present invention. Detailed Implementation

[0033] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0034] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0035] It should be noted that the data in this embodiment are all derived from a comprehensive analysis and evaluation of historical test data and corresponding historical test results from the three months prior to this test. Those skilled in the art will understand that the determination of the above-mentioned parameters for any single item in this invention can be achieved by selecting the value with the highest percentage based on the data distribution as the preset standard parameter, using weighted summation to obtain the value as the preset standard parameter, substituting each historical data point into a specific formula and using the value obtained from that formula as the preset standard parameter, or other selection methods, as long as the invention can clearly define different specific situations in the single-item judgment process through the obtained values.

[0036] It should be noted that the data in this embodiment are all results obtained through preliminary experiments before this test using the method described in this invention. Each preset value can be adjusted according to the specific application, as long as the method described in this invention can clearly define different specific situations in the single-item judgment process through the acquired values. The preset values ​​set in this embodiment are all obtained from preliminary experiments, including the correction coefficients, which were also selected through experimental verification.

[0037] Please see Figures 1 to 5The figures shown are, respectively, a flowchart of the protein composition analysis method for succinyl gelatin injection according to an embodiment of the present invention; a schematic diagram of the protein percentage distribution 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 flowchart of the qualification of the identification process based on the protein component consistency index according to an embodiment of the present invention.

[0038] The protein composition analysis method for succinyl gelatin injection according to embodiments of the present invention includes:

[0039] Step S1: Prepare the chromatographic mobile phase and several reagents respectively;

[0040] Step S2: Pre-treat several succinyl gelatin injection samples to obtain several peptide fragments.

[0041] Step S3: Set the chromatographic and mass spectrometric conditions;

[0042] Step S4: After separating the peptides by chromatograph, elute them according to the elution program;

[0043] Step S5: Use a mass spectrometer to collect data of the eluted peptides to obtain primary and secondary mass spectra;

[0044] Step S6: The obtained mass spectrum is compared with the database using software to identify the protein composition in the sample.

[0045] Step S7: Determine the qualification of the identification process based on the protein component consistency index. If the process is deemed unqualified, increase the number of samples based on the difference between the preset consistency index and the protein component consistency index.

[0046] Specifically, the chromatographic mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.1% formic acid-water solution and mobile phase B is a 0.1% formic acid-acetonitrile solution.

[0047] Specifically, the preparation process of mobile phase A includes:

[0048] Measure 1000 mL of pure water into the mobile phase bottle using a graduated cylinder, then add 1 mL of formic acid using a pipette, mix well, and you will get mobile phase A.

[0049] The preparation process of mobile phase B includes:

[0050] Measure 1000 mL of acetonitrile into the mobile phase bottle using a graduated cylinder, then add 1 mL of formic acid using a pipette and mix well to obtain mobile phase B.

[0051] Specifically, the reagents include NH4HCO3 solution, DTT solution, IAM solution, Rapidest SF (0.1%) solution and Trypsin solution.

[0052] Specifically, the preparation process of each reagent includes:

[0053] 50 mM NH4HCO3 solution: Weigh 20.12 mg of NH4HCO3 into a 15 mL centrifuge tube, add 5 mL of pure water, mix well, and the solution is ready.

[0054] 500 mM DTT solution: Weigh 40.07 mg DTT into a 1.5 mL centrifuge tube, add 256 μL of pure water using a pipette, mix well, and you will get a DTT concentration of approximately 1 M; add 100 μL of 1 M DTT to 100 μL of pure water using a pipette, mix well, and you will get the solution.

[0055] 500 mM IAM solution: Weigh 92.25 mg IAM into a 1.5 mL centrifuge tube, add 500 μL of pure water using a pipette, mix well, and you will get IAM with a concentration of approximately 1 M; add 100 μL of 1 M IAM to 100 μL of pure water using a pipette, mix well, and you will get the solution.

[0056] Rapigest SF (0.1%) solution: Take one vial of 1 mg Rapigest SF lyophilized powder, add 1000 µL of pure water, and mix by pipetting 5-10 times with a pipette tip.

[0057] 0.5 µg / µL Trypsin solution: Take one 25 µg vial of Trypsin lyophilized powder, add 50 µL of pure water, and mix by pipetting 5-10 times with a pipette tip.

[0058] 10% Formic Acid Aqueous Solution: Use a pipette to measure 100 µL of formic acid, add 900 µL of pure water, mix well, and the solution is ready.

[0059] Specifically, the pretreatment process includes: pretreatment, denaturation, reduction, alkylation, termination of alkylation, enzyme digestion and quenching.

[0060] Specifically, the pretreatment process for each sample, involving the selection of a predetermined number of succinyl gelatin injection samples, includes:

[0061] Pretreatment: Measure 375 µL of succinyl gelatin injection solution into a 1.5 mL EP tube, add 625 µL of 50 mM NH4HCO3 solution, and heat at 80 °C for 10 min;

[0062] Denaturation: Take 50 µL of the pretreated sample, add 50 µL of Rapigest SF surfactant (0.1%), mix, and heat at 80 °C for 10 min;

[0063] Reduction: Add 2 µL of 500 mM DTT solution (final concentration approximately 10 mM) to the denatured solution and react at 60 °C for 37 min;

[0064] Alkylation: Add 4 µL of 500 mM IAM solution (final concentration approximately 20 mM) to the reduction solution and react at room temperature in the dark for 30 min.

[0065] Termination of alkylation: Add 2 µL of 500 mM DTT solution to the alkylation solution and mix well;

[0066] Enzymatic digestion: Add 10 µL of 0.5 µg / µL Trypsin to the alkylation termination solution and digest overnight at 37°C for 12 h;

[0067] Quenching: Add 10 µL of 10% formic acid solution to the enzyme digestion solution, vortex for 30 s, let stand at room temperature for 30 min, then centrifuge (14,000 rpm, 10 min, 4 ℃), and transfer the supernatant to a sample vial for LC-MS / MS analysis.

[0068] In this embodiment of the invention, the preset quantity is 9, but this value is not limited to this. Those skilled in the art can adjust this value according to actual needs.

[0069] Specifically, the chromatographic conditions included: column: ACQUITY™ 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.

[0070] Specifically, the chromatographic system used was the Vanquish™ Flex UHPLC system.

[0071] Specifically, the elution procedure is shown in Table 1.

[0072] Table 1. Elution Procedure Flowchart

[0073]

[0074] Specifically, the mass spectrometry conditions included: ion source: electrospray ionization 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℃; auxiliary gas heater temperature: 280℃; resolution: Full MS 70,000 FWHM, ddMS2 17,500 FWHM; scan range: 200 m / z–2000 m / z.

[0075] Specifically, Full MS is the primary mass spectrum, and ddMS2 is the secondary mass spectrum.

[0076] Specifically, the mass spectrometer used is the Q Exactive Plus mass spectrometer.

[0077] Specifically, the mass spectrometry parameters also include: automatic gain control target, 3e6 for stage I; 1e5 for stage II; maximum feed time: 100 ms for stage I; 200 ms for stage II.

[0078] Specifically, the qualification of the identification process is determined based on the protein component 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 according to the difference between the preset consistency index and the protein component consistency index.

[0079] If the protein component consistency index is greater than or equal to the preset consistency index, the identification process is deemed qualified, and the sample protein composition information is recorded.

[0080] In this embodiment of the invention, the preset consistency index is set to 0.9, but this value is not limited to this. Those skilled in the art can adjust the value according to actual needs.

[0081] Specifically, when the protein composition consistency index is lower than the preset threshold, the system automatically determines that the identification is unqualified and dynamically adjusts the number of samples according to the difference, effectively reducing false negative / false positive results and improving the reliability of the data; the sample size is increased only when the consistency is not up to standard, avoiding blindly expanding the scale of testing, saving time and costs, while ensuring statistical power; the protein composition information of qualified samples is systematically recorded, providing a standardized data basis for subsequent analysis and improving the reproducibility of the study.

[0082] Specifically, the number of samples is positively correlated with the difference in the consistency index. If the difference in the consistency index is less than the first preset difference, the number of samples is increased to the corresponding value using the first adjustment coefficient of 1.3.

[0083] If the consistency index difference is greater than or equal to the first preset difference and less than the second preset difference, then the number of samples is increased to the corresponding value using the second adjustment coefficient of 1.6.

[0084] 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 the third adjustment coefficient 2.2;

[0085] The consistency index difference is the difference between the preset consistency index and the protein component consistency index.

[0086] In this embodiment of the invention, the first preset difference is 0.12 and the second preset difference is 0.25. However, the above values ​​are not limited to these values, and those skilled in the art can adjust the above values ​​according to actual needs.

[0087] Specifically, the protein component consistency index is determined by the overlap of protein types and the fluctuation value of protein content. The protein component consistency index = first weighting coefficient × protein type overlap + second weighting coefficient × protein content fluctuation value, wherein the first weighting coefficient is 0.3, the protein type overlap = number of common protein types among samples / total number of protein types identified in all samples, the second weighting coefficient is 0.7, and the protein content fluctuation value = fluctuation threshold / mean of the standard deviation of the percentage content of each protein in the common protein, and the fluctuation threshold is 0.0025.

[0088] Specifically, this invention measures the similarity of protein types between samples by measuring the overlap of protein types, ensuring that key proteins are not missed; and quantifies the expression stability of common proteins by measuring the fluctuation value of protein content, reducing the impact of batch effects or experimental errors.

[0089] The protein composition analysis method of this invention is used to detect the consistency of protein components between different batches of the same product. The protein component consistency index is used to determine the qualification of the identification process, which can avoid errors in the test results due to problems in the analysis method. Such problems include insufficient sample quantity or failure to detect certain proteins due to low content.

[0090] Specifically, in this embodiment of the invention, Proteome Discoverer software is used to search the bovine protein database to identify the protein components in the sample.

[0091] Specifically, the search parameters for the database are shown in Table 2.

[0092] Table 2 Database Search Parameter Table

[0093] item parameter protein library Bovine protein library (Swiss-Prots database, containing 6034 proteins) enzymes trypsin Number of missing cuts 2 Minimum peptide length 4 Fixed embellishment Carbamidomethyl C Variable Modifiers Hydroxy / +15.995 Da (K, P)Succinyl / 100.016 (K, Peptide N-terminus) Search database level 1 quality deviation 10 ppm Secondary quality deviation of the search database 0.02 Da

[0094] Specifically, the succinyl gelatin injection samples in this embodiment were obtained from Jilin Changyuan Pharmaceutical Co., Ltd., and the batch numbers of each sample are shown in Table 3.

[0095] Table 3 Sample Batch Number Table

[0096] name batch number Succinyl gelatin injection 035D2023122111 Succinyl gelatin injection 035D2023122121 Succinyl gelatin injection 035D2023122211 Succinyl gelatin injection 035D2024011011 Succinyl gelatin injection 035D2024011111 Succinyl gelatin injection 035D2024011211 Succinyl gelatin injection 035D2024011311 Succinyl gelatin injection 035D2024011811 Succinyl gelatin injection 035D2024011911

[0097] Specifically, Figure 3The sample batch numbers 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.

[0098] In this embodiment of the invention, collagen was screened from the bovine protein library using Proteome Discoverer software. The identified proteins contained at least two characteristic peptides, and a total of 11 collagen subtypes were identified. The collagen subtypes (protein descriptions) are 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), and A6QPB3 (Collagen alpha-1(X) chain). The protein identification percentages in each sample are shown in Table 4 and 29442 (Collagen alpha-1(XVII) chain) and Q29442 (Collagen alpha-4(IV) chain). Figure 2 As shown.

[0099] Specifically, sequence coverage is calculated by dividing the actual number of detected amino acid residues by the total number of amino acid residues (the theoretical number of amino acids in the 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 NNTotal represents the theoretical number of amino acids.

[0100] Table 4. Protein Identification Percentage Results for Each Sample

[0101]

[0102] Table 5. Standard deviation of the percentage content of each protein in the common proteins

[0103]

[0104] In this embodiment of the invention, for all the above samples, the number of common protein types among the samples is 11, and the total number of protein types identified in all samples is 11. Therefore, the protein type overlap is 1. The mean standard deviation of the percentage content of each protein in the common proteins is 0.0026, therefore, the protein content fluctuation value is 0.96. In summary, the protein composition consistency index among the samples in this embodiment is 0.97, indicating that the identification process is qualified, proving that the sample volume in this embodiment meets the standard and the protein composition among the samples is highly consistent.

[0105] Specifically, from Table 4 and Figure 2 As can be seen, the protein composition results of each batch of succinyl gelatin injection show that the relative content fluctuation range of each component is less than ±3%, proving the consistency of protein composition in samples from different batches. Furthermore, the low volatility of the test results reflects the stability of the protein composition analysis method for succinyl gelatin injection according to the embodiments of the present invention. If the analytical method has systematic or random errors, the test results of different batches of samples will show significant differences. The actual measured fluctuation of ±3% demonstrates the excellent repeatability and reproducibility of the method.

[0106] Specifically, the embodiments of the present invention achieve high-precision and high-sensitivity detection of the complex protein composition of succinyl 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.

[0107] The technical solution of the present invention has been described above with reference to 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 can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

[0108] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for protein composition analysis of succinyl gelatin injection, characterized in that, include: Prepare the chromatographic mobile phase and several reagents respectively; Several peptide fragments were obtained by pretreatment of several succinyl gelatin injection samples. Set chromatographic and mass spectrometric conditions; After separating the peptides by chromatography, elution was performed according to the elution program; Mass spectrometry was used to acquire data of the eluted peptides to obtain primary and secondary mass spectra. The obtained mass spectra were compared with the bovine protein database to identify the protein composition in the sample. The qualification of the identification process is determined by the protein component consistency index, which is determined based on the consistency of protein types and the fluctuation of protein content. If the qualification is not qualified, the number of samples is increased and mass spectra are re-acquired based on the difference between the preset consistency index and the protein component consistency index in order to redetermine the protein composition. The protein composition consistency index is determined by the overlap of protein types and the fluctuation of protein content. The protein composition consistency index = first weighting coefficient × protein type overlap + second weighting coefficient × protein content fluctuation, where protein type overlap = number of common protein types among samples / total number of protein types identified in all samples, and protein content fluctuation = fluctuation threshold / mean of the standard deviation of the percentage content of each protein in the common proteins.

2. The method for protein composition analysis of succinyl 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 a 0.1% formic acid-water solution and mobile phase B is a 0.1% formic acid-acetonitrile solution.

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

4. The method for protein composition analysis of succinyl 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 method for protein composition analysis of succinyl gelatin injection according to claim 4, characterized in that, The chromatographic conditions included: column: ACQUITY™ 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 method for protein composition analysis of succinyl gelatin injection according to claim 5, characterized in that, The mass spectrometry conditions included: ion source: electrospray ionization 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℃; auxiliary gas heater temperature: 280℃; resolution: Full MS 70,000 FWHM, ddMS2 17,500 FWHM; scan range: 200 m / z-2000 m / z.

7. The method for protein composition analysis of succinyl gelatin injection according to claim 6, characterized in that, The qualification of the identification process is determined based on the protein component consistency index, among which, 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 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 deemed qualified, and the sample protein composition information is recorded.

8. The method for protein composition analysis of succinyl gelatin injection according to claim 7, characterized in that, 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.

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

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