A method for peptide mapping detection and analysis of thrombopoietin-mimicking peptides

Abstract

The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting and analyzing the peptide map of thrombopoietin-mimetic peptides, comprising the following steps: (1) The thrombopoietin-mimetic peptides are subjected to buffer system replacement by ultrafiltration centrifugation; (2) Trypsin is used to enzymatically digest the thrombopoietin-mimetic peptides; (3) Reverse-phase high-performance liquid chromatography is used to detect and analyze the enzymatically digested peptide segments. The beneficial effects of the present invention are as follows: In the present invention, sample concentration and buffer replacement are carried out through an ultrafiltration tube, and the sample after enzymatic digestion has high purity and can be directly detected by reverse-phase high-performance liquid chromatography. Therefore, the method for detecting and analyzing the peptide map of thrombopoietin-mimetic peptides is simple to operate, improving the detection efficiency of thrombopoietin-mimetic peptide drugs; in the present invention, by controlling the chromatographic conditions, the accuracy and precision of high-performance liquid chromatography detection are improved, enabling the quality analysis and control of thrombopoietin-mimetic peptide drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting and analyzing the peptide map of thrombopoietin mimetic peptides. Background Art

[0002] Thrombopoietin mimetic peptides are thrombopoietin (TPO) receptor agonists that can significantly increase platelet levels and reduce the bleeding risk by specifically binding to the TPO receptor, activating cell pathways, stimulating the proliferation, differentiation, and maturation of megakaryocytes, and promoting platelet production.

[0003] A peptide map is formed by using a specific proteolytic enzyme to cleave protein polypeptides into small fragments at specific sites according to the amino acid composition characteristics of the protein, and then forming a characteristic fingerprint through certain separation and detection methods (such as high-performance liquid chromatography). With the continuous development of the biopharmaceutical field, peptide map detection has become a key method in the characterization of biopharmaceutical proteins, ensuring the highly specific characterization of biopharmaceutical protein structures. In current traditional recombinant protein peptide map detection methods, the buffer system is replaced by membrane dialysis, which has a high time cost, complex operation, and introduces more impurities during the dialysis process. Therefore, how to design a peptide map detection method for thrombopoietin mimetic peptides that can simply and efficiently obtain a protease-digested sample with high purity while saving time cost has become an urgent problem to be solved. Summary of the Invention

[0004] The present invention provides a method for detecting and analyzing the peptide map of thrombopoietin mimetic peptides in view of the above problems.

[0005] The present invention relates to a method for detecting and analyzing the peptide map of thrombopoietin mimetic peptides, comprising the following steps:

[0006] (1) The buffer system of the thrombopoietin mimetic peptide is replaced by ultrafiltration centrifugation;

[0007] (2) Trypsin is used to digest the thrombopoietin mimetic peptide;

[0008] (3) Reverse-phase high-performance liquid chromatography is used to detect and analyze the digested peptide segments.

[0009] Preferably, in step (1), the test sample and the reference sample are respectively diluted to 1.0 mg / ml with a 1% ammonium bicarbonate solution by mass percentage concentration to obtain a test sample dilution and a reference sample dilution; an appropriate amount of blank buffer is taken and diluted with a 1% ammonium bicarbonate solution by the same multiple as the test sample to obtain a blank dilution;

[0010] Take the test sample diluent, reference diluent, and blank diluent, and add them to a 10 kD ultrafiltration tube respectively, then centrifuge for 30 min; after centrifugation, discard the lower filtrate, add ammonium bicarbonate solution with a mass percentage concentration of 1% to the 10 kD ultrafiltration tube, centrifuge for 30 min, and repeat the above centrifugation operation twice; rinse the filter membranes on both sides of the ultrafiltration tube with ammonium bicarbonate solution with a mass percentage concentration of 1%, and collect the washing solution in the ultrafiltration sleeve to obtain the test sample solution, reference solution, and blank solution.

[0011] Preferably, in step (2), take the test sample solution, reference solution, and blank solution in the ultrafiltration sleeve, add trypsin solution with a mass concentration of 0.5 mg / ml respectively, and carry out enzymatic hydrolysis reaction at 37°C; after the enzymatic digestion is completed, add acetic acid solution with a mass percentage concentration of 50% to terminate the enzymatic digestion respectively, centrifuge and collect the supernatant to obtain the test sample supernatant, reference supernatant, and blank supernatant.

[0012] Preferably, the volume ratio of the test sample solution, reference solution, blank solution to the trypsin solution is 10:1, and the volume ratio of the test sample solution, reference solution, blank solution to the acetic acid solution is 10:1.

[0013] Preferably, step (3) includes sample preparation, reverse-phase high-performance liquid chromatography detection, and experimental result analysis; in the reverse-phase high-performance liquid chromatography detection, the chromatographic conditions are as follows:

[0014] Chromatographic column: Kromasil 300-5-C18 chromatographic column, 4.6×250 mm, 5 μm;

[0015] Guard column: Kromasil 300-5-C18 3.0-4.6 mm guard starter kit;

[0016] Mobile phase A: 0.1% trifluoroacetic acid - aqueous solution;

[0017] Mobile phase B: 0.1% trifluoroacetic acid - acetonitrile solution;

[0018] Mobile phase C: 70% acetonitrile - aqueous solution;

[0019] Detection wavelength: 214 nm;

[0020] Injection volume: 50 μl;

[0021] Flow rate: 1.0 ml / min;

[0022] Column temperature: 35°C;

[0023] Sample tray temperature: 4°C;

[0024] Elution mode: gradient elution.

[0025] Preferably, the procedure of the gradient elution is:

[0026] .

[0027] Preferably, in the sample configuration, the blank supernatant is taken as the enzyme digestion blank solution; the control supernatant is taken to prepare three parallel samples, which are recorded as enzyme digestion control solution 1, enzyme digestion control solution 2 and enzyme digestion control solution 3; the test sample supernatant is taken as the enzyme digestion test sample solution; the same volume of the control sample supernatant and the test sample supernatant are mixed to prepare a 1:1 enzyme digestion control sample-test sample mixed solution.

[0028] Preferably, in the reversed-phase high-performance liquid chromatography detection, the injection order is enzyme digestion blank solution, enzyme digestion reference solution 1, enzyme digestion reference solution 2, enzyme digestion reference solution 3, enzyme digestion test solution and enzyme digestion reference-test solution 1:1 mixed solution.

[0029] Preferably, in the reversed-phase high performance liquid chromatography detection, the integration parameters are:

[0030] Integration algorithm: Apextrack, apex detection; integration interval: 4.5–40; peak width: 10 s; threshold: 300; peak integration: 0.5% of peak start and 0.5% of peak end; minimum height: 0 μV; minimum area: 300,000.

[0031] Preferably, in the experimental result analysis, the identification peak includes identification peak 1, identification peak 2, identification peak 3 and identification peak 4, and the relative retention times of identification peak 1, identification peak 2, identification peak 3 and identification peak 4 are 18.750min±0.5min, 22.897±0.5min, 25.069±0.5min, and 27.100±0.5min, respectively;

[0032] Experimental acceptance criteria: 1) No significant new peaks appear in the peptide map. Criteria for determining significant new peaks are: the new peak area in the peptide map is ≥ 0.5% of the total peak area, and it can also be detected in the peptide map of a 1:1 test: reference solution, with the peak area being 50 ± 20% of that in the peptide map of the test solution; 2) The peak shapes of the identified peaks in enzyme digestion reference solution 1, enzyme digestion reference solution 2, and enzyme digestion reference solution 3 are consistent;

[0033] Determination criteria for test results: 1) The peak shapes of the labeled peaks in the enzyme digestion control solution 1, enzyme digestion control solution 2, enzyme digestion control solution 3, enzyme digestion test sample solution, and enzyme digestion control - test sample 1:1 mixed solution are all the same; 2) Using the labeled peak 4 as the internal standard peak, the relative peak area ratios of each labeled peak need to meet the requirement that the ratio of the relative peak area of the labeled peak of the test sample to the relative peak area of the labeled peak of the control sample should be between 100 ± 15%;

[0034] Formula for relative peak area:

[0035] where Area n is the peak area of the nth peptide peak in the sample, n = 1, 2, 3...

[0036] Area 内标 is the peak area of the internal standard peak.

[0037] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0038] (1) In the present invention, sample concentration and buffer replacement are carried out through an ultrafiltration tube, and the sample after enzymatic digestion has high purity and can be directly detected by reversed - phase high - performance liquid chromatography. Therefore, the operation of the peptide map detection and analysis method for the thrombopoietin mimetic peptide is simple, improving the detection efficiency of thrombopoietin mimetic drugs;

[0039] (2) In the present invention, by controlling the chromatographic conditions, the accuracy and precision of high - performance liquid chromatography detection are improved, enabling the quality analysis and control of thrombopoietin mimetic drugs;

[0040] (3) In the present invention, three enzyme digestion control solutions are set up. Through the system suitability test, it is ensured that the detection experiment is valid, guaranteeing the accurate determination of the thrombopoietin mimetic peptide. Description of the Drawings

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments.

[0042] Figure 1 It is the peptide map of the enzyme digestion blank solution provided in Example 1;

[0043] Figure 2 It is the overlapping peptide map of the enzyme digestion control solutions 1, 2, and 3 provided in Example 1;

[0044] Figure 3 It is the peptide map of the enzyme digestion test sample solution provided in Example 1;

[0045] Figure 4 It is the peptide map of the enzyme digestion control - test sample 1:1 mixed solution provided in Example 1;

[0046] Figure 5 Overlapping peptide maps of the restriction digestion control 1, restriction digestion control solution 2, restriction digestion control solution 3, restriction digestion test solution, and restriction digestion control - test article 1:1 mixed solution provided for Example 1. Detailed implementation manners

[0047] In order to more clearly understand the above - mentioned objects, features, and advantages of the present invention, the present invention will be further described below in conjunction with embodiments.

[0048] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may be implemented in other ways different from those described herein. Therefore, the present invention is not limited by the specific embodiments disclosed in the following specification.

[0049] Example 1

[0050] A method for detecting and analyzing the peptide map of a thrombopoietin - mimetic peptide, characterized by comprising the following steps:

[0051] (1) Preparation of solutions:

[0052] 1% ammonium bicarbonate solution: Weigh 1 g of ammonium bicarbonate, dissolve it with ultrapure water, and make up the volume to 100 ml.

[0053] 0.5 mg / ml trypsin solution: Take 20 μg of trypsin, add 40 μl of trypsin buffer solution and dilute it to 0.5 mg / ml.

[0054] Preparation of blank buffer solution: Weigh 1 g of polysorbate 20, dissolve it with purified water, and make up the volume to 100 ml to obtain polysorbate 20 solution; weigh 8 g of mannitol, 4 g of sucrose, and 0.32 g of histidine, adjust the pH to 5.0 with dilute hydrochloric acid, dissolve it with water, add 0.8 ml of the prepared polysorbate 20 solution, mix well, make up the volume to 200 ml, filter it through a 0.22 - μm filter membrane to obtain the blank buffer solution.

[0055] (2) Dilution of sample solutions:

[0056] Dilute the test article and the control with 1% ammonium bicarbonate solution by mass percentage concentration to 1.0 mg / ml respectively to obtain the test article dilution and the control dilution; take an appropriate amount of blank buffer solution, dilute it with 1% ammonium bicarbonate solution by the same multiple as the test article to obtain the blank dilution.

[0057] (3) Ultrafiltration and buffer exchange:

[0058] Take 400 μl of the test sample diluent, reference diluent, and blank diluent respectively, add them into 10 kD ultrafiltration tubes, and centrifuge at 9600 g for 30 min at 4 °C; after centrifugation, discard the lower filtrate, add 200 μl of 1% ammonium bicarbonate solution to the ultrafiltration tubes, centrifuge at 9600 g for 30 min at 4 °C, and repeat the above centrifugation operation twice; take 200 μl of 1% ammonium bicarbonate solution in the ultrafiltration tubes, and rinse the ultrafiltration tube filter membrane 10 times with a pipette (5 times on each side of the filter membrane), then invert the ultrafiltration tube into another centrifuge sleeve, and centrifuge at 1000 g for 3 min at 4 °C; take 1% ammonium bicarbonate solution into the ultrafiltration tube in the previous step in two portions, 100 μl each time, rinse the ultrafiltration tube filter membrane 10 times (5 times on each side of the filter membrane), collect the washing solution in the ultrafiltration sleeve to obtain the test sample solution, reference solution, and blank solution.

[0059] (4)Trypsin digestion of thrombopoietin mimetic peptide:

[0060] Take 400 μl of the test sample solution, reference solution, and blank solution in the ultrafiltration sleeve respectively, add 40 μl of trypsin solution with a mass concentration of 0.5 mg / ml, and incubate in a water bath at 37 °C for 18 h; after the digestion is completed, add 40 μl of acetic acid solution with a mass percentage concentration of 50% to terminate the digestion respectively, centrifuge at 9600 g for 10 min at 4 °C, collect the supernatant to obtain the test sample supernatant, reference supernatant, and blank supernatant.

[0061] (5)Sample preparation:

[0062] Take 200 μl of the blank supernatant as the digestion blank solution; take 110 μl of the reference supernatant each to prepare 3 parallel samples, denoted as digested reference solution 1, digested reference solution 2, and digested reference solution 3; take 200 μl of the test sample supernatant as the digested test sample solution; take 100 μl of the reference supernatant and the test sample supernatant respectively, mix them evenly as the 1:1 mixed solution of digested reference - test sample.

[0063] (6)Preparation of the mobile phase for high - performance liquid chromatography:

[0064] Mobile phase A: Pipette 2.0 ml of TFA into 2000 ml of ultrapure water, mix well, and perform ultrasonic degassing for 20 min.

[0065] Mobile phase B: Pipette 1.0 ml of TFA into 1000 ml of acetonitrile (chromatographic grade), mix well, and perform ultrasonic degassing for 20 min.

[0066] Mobile phase C: Measure 350 ml of acetonitrile (chromatographic grade) and 150 ml of ultrapure water, mix well, and perform ultrasonic degassing for 20 min.

[0067] (7)Equilibration and flushing before injection of high - performance liquid chromatography:

[0068] Rinse the tubing with mobile phases A, B, and C respectively. After rinsing the injection valve, install the chromatographic column at a low flow rate according to the flow direction marked on the column. First, rinse with 100% mobile phase B at a flow rate of 1.0 ml / min for 10 column volumes, then rinse with 100% mobile phase A at a flow rate of 1.0 m / min for 10 column volumes, and finally equilibrate the chromatographic column with 98% mobile phase A and 2% mobile phase B at a flow rate of 1.0 ml / min for 20 column volumes.

[0069] (8) The chromatographic conditions for high performance liquid chromatography are as follows:

[0070] The model of the high performance liquid chromatograph is: Waters e2695;

[0071] Chromatographic column: Kromasil 300 - 5 - C18 chromatographic column, 4.6×250 mm, 5 μm;

[0072] Guard column: Kromasil 300 - 5 - C18 3.0 - 4.6 mm guard starter kit;

[0073] Mobile phase A: 0.1% trifluoroacetic acid - aqueous solution;

[0074] Mobile phase B: 0.1% trifluoroacetic acid - acetonitrile solution;

[0075] Mobile phase C: 70% acetonitrile - aqueous solution;

[0076] Detection wavelength: 214 nm;

[0077] Injection volume: 50 μl;

[0078] Flow rate: 1.0 ml / min;

[0079] Column temperature: 35 °C;

[0080] Sample tray temperature: 4 °C;

[0081] Elution mode: gradient elution.

[0082] The program for gradient elution is as follows:

[0083] 。

[0084] (9) Edit the batch analysis table for high performance liquid chromatography:

[0085] Input the sample vial number, sample name, injection volume, and injection sequence; the injection sequence is: inject one needle of mobile phase (mobile phase A: mobile phase B = 98:2), one needle of digested blank solution, one needle of digested reference solution 1, one needle of digested reference solution 2, one needle of digested reference solution 3, one needle of digested test sample solution, and one needle of digested reference - test sample 1:1 mixed solution.

[0086] (10)Flushing after injection in high - performance liquid chromatography:

[0087] Flush the chromatographic column with 100% mobile phase C at a flow rate of 1.0 ml / min for 30 min.

[0088] (11)Integration parameters of high - performance liquid chromatography:

[0089] Integration algorithm: Apextrack, vertex detection; integration range: 4.5 - 40; peak width: 10; threshold: 300; peak integration: 0.5% at the peak start percentage and 0.5% at the peak end percentage; minimum height: 0 μV; minimum area: 300000.

[0090] (12)Analysis of experimental results:

[0091] The peptide map of the digested blank solution is shown in Figure 1 ; the overlapping peptide maps of the digested reference solutions 1, 2, and 3 are shown in Figure 2 , and the chromatographic peak information is shown in Table 1; the peptide map of the digested test sample solution is shown in Figure 3 ; the peptide map of the digested reference - test sample 1:1 mixed solution is shown in Figure 4 ; the overlapping peptide maps of the digested reference 1, digested reference solution 2, digested reference solution 3, digested test sample solution, and digested reference - test sample 1:1 mixed solution are shown in Figure 5 , and the chromatographic peak information is shown in Table 2; the results of the relative peak area ratios of each identified peak in the digested test sample solution are shown in Table 3.

[0092] Table 1. Chromatographic peak results of the overlapping peptide maps of the digested reference solutions 1, 2, and 3

[0093]

[0094] Table 2. Chromatographic peak results of the overlapping peptide maps of the digested reference, test sample, and reference - test sample 1:1 mixed solutions

[0095]

[0096] Table 3. Results of the relative peak area ratios of each identified peak in the digested test sample solution

[0097]

[0098] From Figures 1-5It can be obtained from Table 1-3 that the peak shapes of the identified peaks in the peptide map are consistent, and no significant new peaks appear. The relative peak area ratios of the identified peaks are all between 100±15%, indicating that the test samples of this batch meet the detection standards.

[0099] As described above, it is only the preferred embodiment of the present invention, and it is not a limitation to the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes and apply them to other fields. However, as long as it does not depart from the technical solution content of the present invention, any simple modification and equivalent change made to the above embodiments according to the technical essence of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A method for detecting and analyzing the peptide map of a thrombopoietin mimetic peptide, characterized in that, It includes the following steps: (1) The thrombopoietin mimetic peptide is subjected to buffer system replacement by ultrafiltration centrifugation; (2) The thrombopoietin mimetic peptide is enzymatically hydrolyzed with trypsin; (3) The enzymatically hydrolyzed peptide segments are detected and analyzed by reversed-phase high performance liquid chromatography; Step (3) includes sample preparation, reversed-phase high performance liquid chromatography detection and experimental result analysis; In the reversed-phase high performance liquid chromatography detection, the chromatographic conditions are: Chromatographic column: Kromasil 300-5-C18 chromatographic column, 4.6×250mm, 5μm; Guard column: Kromasil 300-5-C18 3.0-4.6mm guard starter kit; Mobile phase A: 0.1% trifluoroacetic acid-aqueous solution; Mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution; Mobile phase C: 70% acetonitrile-aqueous solution; Detection wavelength: 214nm; Elution mode: gradient elution; The program of the gradient elution is: In the experimental result analysis, the identified peaks include identified peak 1, identified peak 2, identified peak 3 and identified peak 4, and the retention times of the identified peak 1, identified peak 2, identified peak 3 and identified peak 4 are 18.750min±0.5min, 22.897±0.5min, 25.069±0.5min, 27.100±0.5min respectively.

2. The method for peptide mapping detection and analysis of thrombopoietin mimetic peptides according to claim 1, characterized in that In step (1), the test sample and the reference sample are respectively diluted to 1.0mg / ml with a 1% ammonium bicarbonate solution by mass percentage concentration to obtain a test sample dilution and a reference sample dilution; An appropriate amount of blank buffer is taken and diluted with a 1% ammonium bicarbonate solution by the same multiple as the test sample to obtain a blank dilution; The test sample dilution, the reference sample dilution and the blank dilution are taken and respectively added into a 10kD ultrafiltration tube, and centrifuged for 30min; After centrifugation, the lower layer filtrate is discarded, and a 1% ammonium bicarbonate solution by mass percentage concentration is added to the 10kD ultrafiltration tube, and centrifuged for 30min, and the above centrifugation operation is repeated twice; The two-side filter membranes of the ultrafiltration tube are rinsed with a 1% ammonium bicarbonate solution by mass percentage concentration, and the washing liquid is collected in the ultrafiltration sleeve to obtain a test sample solution, a reference sample solution and a blank solution.

3. The method for peptide mapping detection and analysis of thrombopoietin mimetic peptide according to claim 2, wherein In step (2), the test sample solution, the reference sample solution and the blank solution in the ultrafiltration sleeve are respectively added with a trypsin solution with a mass concentration of 0.5mg / ml, and an enzymatic hydrolysis reaction is carried out at 37°C; After the enzymatic digestion is completed, a 50% acetic acid solution by mass percentage concentration is respectively added to terminate the enzymatic digestion, centrifuged and the supernatant is collected to obtain a test sample supernatant, a reference sample supernatant and a blank supernatant.

4. The method for peptide mapping detection and analysis of thrombopoietin-mimicking peptide according to claim 3, wherein The volume ratios of the test sample solution, the reference sample solution, the blank solution to the trypsin solution are all 10:1, and the volume ratios of the test sample solution, the reference sample solution, the blank solution to the acetic acid solution are all 10:

1.

5. The method for peptide mapping detection and analysis of thrombopoietin-mimicking peptides according to claim 3, wherein In the reversed-phase high performance liquid chromatography detection, the injection volume: 50μl; Flow rate: 1.0ml / min; Column temperature: 35°C; Sample tray temperature: 4°C.

6. The method for detecting and analyzing the peptide map of the thrombopoietin mimetic peptide according to claim 5, characterized in that, In the sample preparation, the blank supernatant is taken as the digestion blank solution; the supernatant of the reference substance is taken to prepare 3 parallel samples, denoted as digestion reference substance solution 1, digestion reference substance solution 2, and digestion reference substance solution 3; the supernatant of the test sample is taken as the digestion test sample solution; the supernatant of the reference substance and the supernatant of the test sample with the same volume are mixed to serve as the digestion reference substance-test sample 1:1 mixed solution.

7. The method for peptide mapping detection and analysis of thrombopoietin-mimicking peptide according to claim 6, characterized in that, In the reversed-phase high performance liquid chromatography detection, the injection sequence is successively the digestion blank solution, digestion reference substance solution 1, digestion reference substance solution 2, digestion reference substance solution 3, digestion test sample solution, and digestion reference substance-test sample 1:1 mixed solution.

8. The method for detecting and analyzing the peptide map of the thrombopoietin mimetic peptide according to claim 5, characterized in that, In the reversed-phase high performance liquid chromatography detection, the integration parameters are as follows: integration algorithm: Apextrack, apex detection; integration interval: 4.5 - 40; peak width: 10 s; threshold: 300; peak integration: the peak start percentage is 0.5%, and the peak end percentage is 0.5%; minimum height: 0 μV; minimum area: 300000.

9. The peptide map detection and analysis method of the thrombopoietin mimetic peptide according to claim 6, characterized in that The standard for the experiment to be valid: 1) No significant new peaks appear in the peptide map results. The determination standard for significant new peaks is: the peak area of the new peak in the peptide map ≥ 0.5% of the total peak area, and it can also be detected in the peptide map of the test sample:reference substance = 1:1 mixed solution, and the peak area of this peak is 50 ± 20% of the peptide map of the test sample solution; 2) The peak shapes of the respective labeled peaks of digestion reference substance solution 1, digestion reference substance solution 2, and digestion reference substance solution 3 are all the same; The determination standard for the detection results: 1) The peak shapes of the labeled peaks of digestion reference substance solution 1, digestion reference substance solution 2, digestion reference substance solution 3, digestion test sample solution, and digestion reference substance-test sample 1:1 mixed solution are all the same; 2) Taking the labeled peak 4 as the internal standard peak, the relative peak area ratios of the respective labeled peaks need to meet: the ratio of the relative peak area of the labeled peak of the test sample to the relative peak area of the labeled peak of the reference substance should be between 100 ± 15%; the calculation formula for the relative peak area: RArea= , where Area n is the peak area of the n-th peptide peak in the sample, n = 1, 2, 3...; Area 内标 It is the peak area of the internal standard peak.

Images