Method for analyzing iodination modification level and modification site of protein

Abstract

The invention relates to the technical field of biology, in particular to an analysis method for analyzing the iodination modification level of protein in a biological sample and identifying modification sites of the protein. Target protein to be detected is extracted from a biological sample to be detected through co-immunoprecipitation, the content of iodinated tyrosine in the protein is detected through acid hydrolysis, and the iodinated modification level of the protein to be detected is obtained. The iodination site information of the target protein is obtained by performing iodination chemical modification on the target protein by using an iodinated salt-Fenton reaction, then performing enzymolysis digestion, and analyzing the digested peptide fragment by using a high-resolution mass spectrum. The method disclosed by the invention can be used for accurately and quantitatively analyzing the iodination modification peptide fragment and analyzing the iodination modification level of the iodination modification peptide fragment, has the advantages of high sensitivity and simplicity and convenience in use, and provides a good technical means for iodination modification of protein / peptide.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an analysis method for analyzing the iodination modification level of proteins in biological samples and identifying the modification sites thereof. Background Art

[0002] The study of protein modification has received great attention in the process of protein function research. Common protein modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, etc. By adding modifying groups to one or more amino acid residues of a protein, the physical and chemical properties of the protein can be changed, thereby affecting the spatial conformation, activity, localization, and interaction between proteins. Protein modification plays a key role in a variety of cellular processes. Protein phosphorylation modification is often closely related to cell signal transduction, ubiquitination modification is related to protein degradation, methylation is related to protein transcription and translation, and protein glycosylation is related to the pathological changes of diabetes and tumors. The present invention provides for the first time a method for analyzing the iodination modification level of a protein and its modification sites. It provides a technical means for exploring protein iodination modification and biological functions. CN202110047611.1 discloses using ultraviolet light generated by an ultraviolet light source to directly act on a protein solution containing a water-soluble chloride salt, bromide salt or iodide salt, thereby directly halogenating aromatic amino acids, avoiding various complex catalysts, ligands and harsh reaction conditions in conventional aromatic amino acid halogenation reaction systems. However, the halogenation modification method and modification efficiency of proteins, the detection of iodination modification of proteins, and the detection of iodination modification levels of proteins need to be further developed and improved. Summary of the invention

[0003] The purpose of the present application is to provide a method for analyzing the iodination modification level and modification site of proteins in biological samples.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides a method for analyzing the iodination modification level of a protein, the method comprising: acid hydrolyzing the target protein, adjusting the pH to alkaline, derivatizing the hydrolyzate, and detecting and determining the content of iodinated tyrosine in the hydrolyzed protein, wherein the content of iodinated tyrosine is the iodination modification level of the target protein.

[0006] In some embodiments, the method for analyzing the iodination modification level of a protein includes: acid hydrolyzing the target protein into single amino acids, adjusting the pH to alkaline, and then treating it with a derivatization reagent, and detecting and determining the content of mono-iodinated tyrosine and di-iodinated tyrosine therein after the derivatization treatment, wherein the content of iodinated tyrosine is the iodination modification level of the target protein.

[0007] In the present invention, preferably, the derivatization treatment is to use SPTPP ((5-N-succinimidoxy-5-oxopentyl)-triphenylphosphonium bromide) to derivatize the hydrolyzate. Ultra-high performance liquid chromatography-quadrupole mass spectrometry is used to evaluate the iodination modification level of the target protein.

[0008] In some embodiments, the target protein is extracted from the biological sample to be tested. Preferably, the target protein is extracted from the biological sample to be tested by immunoprecipitation.

[0009] In some embodiments, the acid used in the acid hydrolysis is a strong acid; preferably, the strong acid is hydrochloric acid.

[0010] In some embodiments, the acid hydrolysis is performed using hydrochloric acid. Preferably, the hydrochloric acid concentration of the hydrochloric acid hydrolysis is 6N, the volume ratio of the target protein solution to the hydrochloric acid is 1:1, the acid hydrolysis temperature is 110°C, and the acid hydrolysis time is 20 hours; after the acid hydrolysis, the pH of the sample is adjusted to 8.0 with sodium hydroxide, and mass spectrometry analysis is performed after SPTPP derivatization.

[0011] In some embodiments, an ultra-high performance liquid chromatography-quadrupole mass spectrometer is used to evaluate the iodination modification level of the target protein, wherein: (1) UPLC liquid phase conditions: the chromatographic column is Poroshell HPH-C18, and the column length is 100 mm; the mobile phase gradient is methanol (A) and ultrapure water (B), and the gradient changes are 0-1 min, 30% A; 1-15 min, 30%-60% A; 15-17 min, 60%-75% A; 17-18 min, 75%-100% A; 18-26 min, 100% A; 26.1-30 min, 30% A; the mobile phase flow rate is 0.3 mL / min; the column temperature is 35°C, and the injection volume is 2-10 μl. (2) Mass spectrometry conditions: ion source: ESI source; data scanning mode: positive ion mode; desolvation gas temperature: 500°C; capillary voltage: 3 kV; desolvation gas flow rate: 1000 L / Hr; source temperature: 150°C; measurement method: selected reaction monitoring (MRM).

[0012] In a second aspect of the present invention, the present invention provides a method for analyzing protein iodination sites, the method comprising:

[0013] (4) using the iodine salt-Fenton reaction to chemically modify the target protein by iodination;

[0014] (5) digesting the chemically modified target protein with an enzyme and enriching the digested peptides;

[0015] (6) Performing high-resolution mass spectrometry data acquisition on the target protein peptides to be detected, analyzing the acquired mass spectrometry information, and obtaining the iodination modification site information of the target protein.

[0016] Preferably, in the present invention, the iodination chemical modification is iodination modification of tyrosine.

[0017] In some embodiments, the iodine salt is sodium iodide.

[0018] In some embodiments, the target protein is extracted from a biological sample to be tested. Preferably, the biological sample is a cell sample or a tissue sample.

[0019] In the present invention, the biological sample can be subjected to one or more of the following methods: crushing, lysis, centrifugation, and chromatography to release and separate the target protein from the biological sample. The crushing method includes one or more of the following methods: liquid nitrogen freezing crushing, ultrasonic crushing, tissue cell extractor crushing, and repeated freeze-thaw crushing.

[0020] In some embodiments, the biological sample to be tested is lysed in a cell lysis solution and then ultrasonically disrupted to release proteins in the cells into the cell lysis solution.

[0021] Preferably, in order to ensure that the target protein is not hydrolyzed by proteases in the environment during the extraction process, protease inhibitors and phosphorylase inhibitors are added to the cell lysate.

[0022] Preferably, during the ultrasonic disruption process, the sample is subjected to ultrasonication under low temperature conditions to prevent protein denaturation or degradation during the cell ultrasonic disruption process.

[0023] In some embodiments, the target protein is extracted from the lysed biological sample to be tested by co-immunoprecipitation.

[0024] In some embodiments, the total protein concentration extracted from the biological sample to be tested is not less than 1 mg / ml protein concentration.

[0025] In some embodiments, the protein chemical iodination modification step includes: reacting the obtained target protein with sodium iodide in a hydrogen peroxide environment via ferrous ion catalysis. The iodination modification can iodine-modify the iodinated tyrosine in the protein, thereby enhancing the probability of identifying the iodinated modification site during mass spectrometry analysis.

[0026] In some embodiments, the protein iodination modification step of the present invention includes: dissolving the target protein in 50 μL of PBS solution, adding 100 μL of 1M sodium iodide solution, 30 μL of 1.5% hydrogen peroxide solution and 30 μL of 5mM ferrous sulfate solution to the target protein.

[0027] In some embodiments, the enzymatic digestion is performed using one or more proteolytic agents selected from the group consisting of trypsin, alkaline endoproteinase, proteinase K, and carboxypeptidase B.

[0028] In some embodiments, high-resolution mass spectrometry data acquisition is to use a tandem mass spectrometer Q-Exactive Orbitrap to analyze the sample in Full-MS-ddMS2 acquisition mode, perform secondary spectrum acquisition on the TOP20 signal, and collect mass spectrometry data for 2 hours. The iodinated protein spectrum data analysis software is commercial Maquant, Proteome discoverer or Mascot.

[0029] The beneficial effects of the present invention are as follows:

[0030] (1) The technology of the present invention adopts mass spectrometry detection and analysis, which can accurately and quantitatively analyze iodinated modified peptides and analyze their iodination modification levels. At the same time, the protein iodination modification technology of the present invention enhances the recognition of iodination sites, and based on the post-derivation technology, it can improve the sensitivity of iodinated peptide analysis and detection, reduce matrix interference in the analysis process, and improve the anti-interference ability of the method, thereby realizing non-targeted protein iodination modification for site identification.

[0031] (2) The present invention provides for the first time a method for analyzing the iodination modification level and modification sites of proteins, providing a technical means for exploring protein iodination modification and function. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the iodinated tyrosine content in Tg protein, NRAS protein, HRAS protein and P53 protein;

[0033] Figure 2 This is the Western-blot result of the sample immunoprecipitation EGFR protein;

[0034] Figure 3 The results are for the identification of iodination modification sites of EGFR protein;

[0035] Figure 4 It is the primary and secondary mass spectrometry information of the high-resolution mass spectrometry of the EEKEY(I) peptide segment;

[0036] Figure 5 The primary and secondary mass spectrometry information of the GAEEKEY(I) peptide segment is high resolution mass spectrometry; DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below.

[0038] Example 1: Detection of iodination modification levels of Tg, NRAS, HRAS and P53 proteins

[0039] 1.1, Acid hydrolysis:

[0040] 1 μg of Tg, NRAS, HRAS and P53 proteins were prepared into 500 μl of protein solution, and then 500 μl of 6N hydrochloric acid was added respectively, and acid hydrolysis was carried out at 110° C. overnight. After acid hydrolysis, the pH was adjusted to 8.0-9.0 with 1 M NaOH.

[0041] 1.2. Derivatization reaction:

[0042] Take 30 μl of PBS solution (derivation reaction buffer solution) with a pH of 8.0 in a 2 mL brown bottle, add the protein acid hydrolyzate of step 1.1, add 20 μl of 30 mM derivatization reagent (SPTPP), seal and vortex to mix, place in a 40°C oven for derivatization reaction for 20 minutes, immediately place it on ice after the reaction, and add 600 μl of 1 M sodium hydroxide (derivation reaction termination solution). After the reaction is terminated, the product is purified and extracted twice with 0.8 mL of ethyl acetate, the ethyl acetate is discarded, and 200 ul of acidification solution is added to the purified derivatization product for acidification, and extracted twice with 0.8 mL of ethyl acetate. The collected upper ethyl acetate extract is blown to near dryness with weak nitrogen gas, 50 μl of methanol is added to rinse the inner wall, vortexed, and the methanol is transferred to the liner tube for UPLC-MS / MS detection.

[0043] 1.3. UPLC-MS / MS detection:

[0044] (1) UPLC liquid phase conditions: the chromatographic column was Poroshell HPH-C18, column length was 100 mm; the mobile phase gradient was methanol (A) and ultrapure water (B), and the gradient changed as follows: 0-1 min, 30% A; 1-15 min, 30%-60% A; 15-17 min, 60%-75% A; 17-18 min, 75%-100% A; 18-26 min, 100% A; 26.1-30 min, 30% A; the mobile phase flow rate was 0.3 mL / min; the column temperature was 35°C, and the injection volume was 2-10 μl.

[0045] (2) Mass spectrometry conditions: ion source: ESI source; data scanning mode: positive ion mode; desolvation gas temperature: 500°C; capillary voltage: 3 kV; desolvation gas flow rate: 1000 L / Hr; source temperature: 150°C; measurement method: selected reaction monitoring mode (MRM). The main mass spectrometry parameters are shown in Table 1 below.

[0046] Table 1: Main mass spectrometry parameters

[0047]

[0048]

[0049] 1.4. Standard curve and minimum detection limit:

[0050] (1) Take a mixed standard solution of MIT and DIT with a concentration of 10 μg / mL, dilute it with pure water, and prepare working solutions with concentration gradients of 0.25 ng / L, 1.00 ng / L, 5.00 ng / L, 20 ng / L, 50 ng / L and 150 ng / L (each working solution contains equal concentrations of MIT and DIT for standby use;

[0051] (2) Take 100 μl of working solution with a concentration gradient of 0.25 ng / L, 1.00 ng / L, 5.00 ng / L, 20 ng / L, 50 ng / L and 150 ng / L, add 50 μl of methanol mixed internal standard solution, vortex and shake to mix, then add 30 μl of derivatization reaction buffer solution and 20 μl of derivatization reagent, seal with a lid and place in a 40°C oven for derivatization reaction for 20 minutes. After the reaction is completed, immediately place it on ice and add 600 μl of stop solution. After the reaction is terminated, use 0.8 mL The product was purified and extracted twice with ethyl acetate, and the ethyl acetate was discarded. 200ul of acidified solution was added to the purified derivative product for acidification, and extracted twice with 0.8mL of ethyl acetate. The collected upper ethyl acetate extract was blown to near dryness with weak nitrogen gas, and 50μl of methanol was added to rinse the inner wall. The methanol was transferred to the liner tube by vortexing. The concentrations of the SPTPP derivative mixed standard working solutions were: 0.5ng / L, 2.00ng / L, 10.00ng / L, 40ng / L, 100ng / L and 300ng / L;

[0052] (3) The SPTPP-derivatized mixed standard working solution was injected with 2 μl for UPLC-MS / MS analysis, and the ratio of the peak area of ​​each standard sample to the peak area of ​​the internal standard (Y) and its concentration (X) were subjected to linear regression analysis to obtain a standard curve.

[0053] The results showed that the concentration of the substance in the range of 0.5 ng / L to 300 ng / L showed a good linear relationship with the peak area of ​​the measured relative internal standard, and the correlation coefficient (γ 2 ) were all greater than 0.99. The detection limits (LOD) of MIT and DIT were calculated based on the signal-to-noise ratio (S / N) and are shown in Table 2 below. The iodination modification levels of the tested Tg, NRAS, HRAS and P53 proteins are shown in Table 2. Figure 1 shown.

[0054] Table 2: MIT and DIT standard curve parameters of the present method

[0055] Serial number Target Linear equations <![CDATA[Correlation coefficient (γ 2 )]]> Detection limit (pg / mL) 1 MIT Y=98.71X-1.84 0.9999 0.07 2 DIT Y=155.24X-0.09 0.9999 0.14

[0056] Example 2: Identification of iodination modification sites of EGFR protein

[0057] 2.1. Protein extraction:

[0058] Take 50 mg of tissue sample and transfer it to 1 ml of cell lysis buffer and appropriate amount of protease inhibitors; use ultrasonic tissue disruptor to break and lyse the tissue cells. Centrifuge the lysed tissue at 10000g centrifugal force at 4℃ for 10 minutes, and the supernatant is the extracted total protein solution.

[0059] 2.2 Protein Quantification

[0060] The protein was quantified using the Bradford method, and the protein concentration in the total protein extract was diluted to 2 mg / mL.

[0061] 2.3. Co-immunoprecipitation:

[0062] Take 500 μl of total protein, add protein magnetic beads A or G to it, incubate at room temperature with shaking for 20 minutes, adsorb the magnetic beads with a magnet and discard the magnetic beads to remove non-specific adsorption proteins of the magnetic beads in the total protein; then add 5 μl of EGFReceptor (D38B1) to the protein Rabbit mAb antibody was added and incubated overnight with shaking. After overnight incubation, protein magnetic beads A were added and incubated at room temperature with shaking for 20 minutes. The magnetic beads were washed 5 times with cell lysis buffer containing protease inhibitors.

[0063] 2.4. Polyacrylamide gel electrophoresis

[0064] The immunoprecipitated protein was added to 30 μl of 1X loading buffer, mixed and heated at 95°C for 5 minutes, the supernatant was added to 3-8% SDS polyacrylamide gel, and electrophoresed at 80V constant voltage for 30 minutes and then at 120V constant voltage for 100 minutes; after electrophoresis, the target protein band was cut off and the protein was recovered using a protein gel recovery kit. In this embodiment, the Solebao PAGE gel protein micro-recovery kit (G7200) was used to recover the protein. The Western-blot results of the sample immunoprecipitated EGFR protein are shown in Figure 1. Figure 2 shown.

[0065] 2.5. Chemical iodination treatment

[0066] The recovered protein was chemically iodinated, and the chemical iodination modification system was as follows: ①140uL PBS, ②50μL target protein (protein concentration not less than 1mg / mL), ③100μL sodium iodide solution, ④30μL 1.5% hydrogen peroxide solution, ⑤30μL 5mM ferrous sulfate solution. After adding the reagents to each group, put it in a 25 degree Celsius incubator at 80 rpm for 1 hour; after the reaction, add 30uL 1M sodium thiosulfate to quench the hydroxyl radicals; then add 500ul frozen methanol to precipitate the protein, centrifuge at 12000rpm at 4℃ for 10 minutes; carefully discard the supernatant; add 400uL ultrapure water, shake and re-dissolve the protein; then add 400uL frozen methanol to precipitate the protein, centrifuge at 12000rpm at 4℃ for 10 minutes; carefully discard the supernatant and wash twice, add 50uL PBS to the precipitated protein to re-dissolve the protein twice and transfer it to a brown vial.

[0067] 2.6 Enzymatic hydrolysis

[0068] The chemically iodinated protein was enzymatically hydrolyzed at a ratio of protein:trypsin = 40:1, and the reaction was carried out in a 37°C water bath overnight. After the enzymatic reaction, a Strata X column (PHENOMENEX, TUBES-10MG / 1ML) was used for desalting and vacuum drying to obtain the enzymatically hydrolyzed peptides.

[0069] 7. Lyophilization and reconstitution

[0070] The desalted enzymatic hydrolysate was freeze-dried and re-dissolved in a 0.1% FA solution.

[0071] 8. Mass spectrometry

[0072] The samples were analyzed using a tandem mass spectrometer Q-Exactive Orbitrap in Full-MS-ddMS2 acquisition mode, and secondary spectra were collected for TOP20 signals, with mass spectrometry data collected for 2 hours.

[0073] (1) Liquid phase separation conditions

[0074] A C18 chromatographic column was used, liquid A was a 0.1% formic acid in water, and liquid B was a 0.1% formic acid in acetonitrile. The liquid phase gradient is shown in Table 3 below.

[0075] Table 3 Liquid phase separation conditions in iodination modification site detection

[0076] Time (min) Flow rate (μL / min) B％ 0 0.3 2 45 0.3 22 60 0.3 35 90 0.3 80 100 0.3 100 105 0.3 2 120 0.3 2

[0077] (2) Mass spectrometry data acquisition parameters

[0078] The Full-MS-ddMS2 acquisition mode was used with a scan range of 100-2000 m / z, 10 parent ions were selected, and the fragmentation energy was 40 V.

[0079] 8. Data Analysis

[0080] The raw files of mass spectrometry test were searched with Proteome Discoverer1.4 software to obtain the corresponding database and finally the protein identification results. The search parameters are as follows:

[0081]

[0082] Test results: Figure 3 The method of this embodiment detects the iodinated modified peptides and site results of the EGFR protein.

[0083] Example 3: Analysis of EEKEY and GAEEKEY iodinated peptides

[0084] 3.1. Derivatization reaction:

[0085] Take 30μl of derivatization reaction buffer solution in a 2mL brown bottle, add the target peptide, add 20μl, 30mM derivatization reagent, seal and vortex to mix, place in a 40℃ oven for derivatization reaction for 20 minutes, immediately put it on ice after the reaction, and add 600μl of derivatization reaction termination solution. After the reaction is terminated, purify and extract the product twice with 0.8mL ethyl acetate, discard the ethyl acetate, add 200ul of acidification solution to the purified derivatization product for acidification, extract it twice with 0.8mL ethyl acetate, collect the upper ethyl acetate extract, blow it to near dryness with weak nitrogen gas, add 50μl methanol to rinse the inner wall, vortex and shake, transfer the methanol to the liner tube, and wait for UPLC-Q-TOF detection.

[0086] 2. Ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry (UPLC-Q-TOF) detection:

[0087] UPLC liquid phase conditions: the chromatographic column is Poroshell HPH-C18, the column length is 100mm; the mobile phase gradient is methanol (A) and ultrapure water (B), and the gradient changes are 0-1min, 30% A; 1-15min, 30%-60% A; 15-17min, 60%-75% A; 17-18min, 75%-100% A; 18-26min, 100% A; 26.1-30min, 30% A; the mobile phase flow rate is 0.3mL / min; the column temperature is 35℃, and the injection volume is 2-10μl. (2) Mass spectrometry conditions: the ion source is: ESI source; the data scanning mode is: positive ion mode; the desolvation temperature is 500℃; the capillary voltage is 3kV; the desolvation flow rate is 800L / Hr; the source temperature is 100℃; the determination method is: MSE.

[0088] Test results: High-resolution primary and secondary mass spectrometry information of EEKEY and GAEEKEY iodinated peptides are as follows: Figure 4 and Figure 5 shown.

[0089] In summary, the present invention provides a method for analyzing the iodination modification level and modification sites of proteins, especially the iodination level and iodination sites of proteins. It includes acid hydrolysis of the target protein, hydrolyzing the target protein into single amino acids, and analyzing the iodination modification level of the target protein by detecting the iodinated tyrosine level; a non-targeted identification method for protein iodination sites is also developed, the target protein is proteolyzed into peptides, and high-resolution mass spectrometry scanning and data analysis are performed to identify all iodination modification sites of the target protein; at the same time, the present invention can also detect specific iodination sites of the target protein, directly analyze the target iodinated peptides produced by proteolysis, and thus obtain the iodination modification sites and levels of the target protein peptides.

[0090] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. A method for analyzing the level of protein iodination modification, characterized in that: The method comprises: acid hydrolyzing the target protein, adjusting the pH to alkaline, derivatizing the hydrolyzate, and detecting and determining the content of iodinated tyrosine in the hydrolyzed protein, wherein the content of iodinated tyrosine is the iodination modification level of the target protein.

2. The method according to claim 1, characterized in that The derivatization treatment is to use a derivatization agent SPTPP to derivatize the hydrolyzate.

3. The method according to claim 2, characterized in that Iodination modification of protein tyrosine; the acid hydrolysis is hydrolysis using a strong acid; preferably, the strong acid is hydrochloric acid.

4. The method according to claim 3, characterized in that: The detection is performed using an ultra-high performance liquid chromatography-quadrupole mass spectrometer, wherein (1) the UPLC liquid phase conditions are: the chromatographic column is Poroshell HPH-C18, column length 100mm; mobile phase gradient: A phase methanol and B phase ultrapure water, the gradient changes are 0-1min, 30%A; 1-15min, 30%-60%A; 15-17min, 60%-75%A; 17-18min, 75%-100%A; 18-26min, 100%A; 26.1-30min, 30%A; mobile phase flow rate is 0.3mL / min; column temperature is 35℃, injection volume is 2-10μl; (2) mass spectrometry conditions: ion source is: ESI source; data scanning mode: positive ion mode; desolvation gas temperature is 500℃; capillary voltage is 3kV; desolvation gas flow rate is 1000L / Hr; source temperature is 150℃; determination method: selected reaction monitoring mode MRM.

5. A method for analyzing protein iodination sites, characterized in that: The method comprises: (1) Using the iodine salt-Fenton reaction to chemically modify the target protein; (2) digesting the chemically modified target protein with an enzyme and enriching the digested peptides; (3) High-resolution mass spectrometry data is collected for the target protein peptides to be detected, and the collected mass spectrometry information is analyzed to obtain the iodination modification site information of the target protein.

6. The method according to claim 5, characterized in that The iodination chemical modification is iodination modification of tyrosine; preferably, the iodine salt is sodium iodide.

7. The method according to claim 7, characterized in that: The step of iodination chemical modification includes: reacting the obtained target protein with sodium iodide in a hydrogen peroxide environment through ferrous ion catalysis to iodine-modify the tyrosine in the protein that can be iodinated.

8. The method according to claim 5, characterized in that Enzymatic digestion is performed using one or more protease digestion reagents selected from the group consisting of trypsin, alkaline endoproteinase, proteinase K, and carboxypeptidase B.

9. The method according to claim 5, characterized in that For high-resolution mass spectrometry data acquisition, the samples were analyzed in Full-MS-ddMS2 acquisition mode using a tandem mass spectrometer Q-Exactive Orbitrap, secondary spectra were acquired for TOP20 signals, and mass spectrometry data were collected for 2 hours.

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