Method for detecting protein pegylation modification sites

Abstract

The application discloses a method for detecting a protein PEG modification site and application thereof. First, a protein is modified by a PEG with a large molecular weight into a peptide segment, without performing parent ion screening, and then the PEG modified peptide segment is directly selected in a part (IDA mode) or all (SWATH mode) with a high primary signal for secondary fragmentation. The interference of PEG fragments is excluded by background deduction, and background specificity deduction is performed on the PEG. Then, the secondary signal of the PEG modified peptide segment is directly analyzed. When the modification site is lysine, trypsin or Lys-C enzyme cannot cut the modified lysine, and by using the characteristics, all the peptide segment sequences formed by the enzyme cutting after the PEG is assumed to be modified can be listed. The actual generated ions are compared one by one, so that the corresponding modified peptide segment can be more accurately matched.

Description

Technical Field

[0001] This invention relates to methods for analyzing proteins after modification, and particularly to a method for detecting protein PEGylation modification sites and its application. Background Technology

[0002] With the development of biotechnology, the production capacity of drugs such as proteins and peptides has been greatly improved. However, the inherent instability, susceptibility to protease degradation, and easy clearance of protein and peptide products in vivo result in short half-lives. Frequent injections can reduce patient compliance and affect treatment efficacy. Molecular modification can prolong half-life. Common molecular modification strategies include increasing the sialic acid content on sugar residues, covalently binding to albumin, and fusing with the Fc region of IgG. Among these, linking to water-soluble PEG molecules has been the most successful. PEG molecules are non-toxic, non-immunogenic, and hydrophilic, and can significantly improve the pharmacokinetic stability of drugs without compromising efficacy. By increasing stability, reducing immunogenicity, and increasing solubility, the half-life of drugs in vivo is prolonged. Since the first PEGylated drug, "Adagen," was approved by the FDA in 1990, more and more biologics have utilized PEGylation to improve molecular half-life. However, detailed structural characterization of PEGylated proteins and peptides still faces significant challenges. For example, the polydispersity of PEG, modification sites, and the number of PEG crosslinks remain important factors.

[0003] When using traditional ESI-MS detection, PEGylation results in a heavily charged molecule, leading to the superposition of spectra of ions with different charge states and PEG fragments, thus affecting the accuracy of resolution due to spectral quality issues. To address these problems, characterization based on MALDI-MS has been widely reported, as MALDI is single-charged and improves spectral complexity. However, for large PEGylated proteins, MALDI provides only limited information due to its limited resolution and ionization efficiency.

[0004] PEG can crosslink with proteins in a site-specific or random manner. In random coupling, PEG molecules with active groups typically couple to the N-terminus or lysine side chain amino groups of the protein. Since proteins have a large number of lysine residues, only a portion of these can couple with PEG. Based on the possible binding regions, PEGylation sites can affect biological activity, and the presence of multiple sites results in a mixture of site isoforms. Therefore, identifying these modification sites is crucial for the characterization of PEGylated drugs.

[0005] Patent document CN113848259A discloses a method and application for detecting protein ubiquitination modification sites based on high-precision mass spectrometry. The method includes the following steps: Step S1: Constructing an in vitro SUMOylation modification reaction system to obtain the target protein; Step S2: Digesting the target protein obtained in Step S1 with trypsin to obtain the digested polypeptide fragments; Step S3: Analyzing the polypeptide obtained in Step S2 using liquid chromatography-mass spectrometry to obtain polypeptide mass spectrometry data, and analyzing the data to obtain the modification sites and the sequence of the core peptide; Step S4: Chemically synthesizing isotope-labeled peptide sequences containing SUMOylation modification sites, confirming the sites using the PRM method, and determining a set of parent-daughter ion pairs; Step S5: Detecting endogenous modification sites using the parent-daughter ion pairs established in Step S4.

[0006] Patent document CN103776909A discloses a method for identifying protein ubiquitination modification sites: the first-stage mass spectrometry uses full mass number scanning, and the second-stage mass spectrometry uses a data-dependent scanning method to analyze the ten parent ions with the highest intensity in the first-stage mass spectrum; the obtained mass spectra are compared with the African Xenopus and human protein sequence database search engines using the Sequest search algorithm to identify peptide sequences, peptide modification sites and their corresponding proteins.

[0007] The aforementioned existing technologies all involve enzymatically digesting the modified protein, screening for precursor ions, determining the modified peptides based on primary mass spectrometry, and then matching the modification sites according to secondary daughter ion data. However, for PEGylated peptides, due to the polydispersity of PEG, instability in ion sources, and the multi-charge characteristics of ESI, it is difficult to determine the modified peptides using primary mass spectrometry. Therefore, the above methods are not suitable for the analysis of PEGylated peptides. Summary of the Invention

[0008] To overcome the shortcomings of existing technologies, this invention aims to provide a method for detecting protein PEGylation modification sites and its application, which can obtain more direct information on protein modification sites and accurately determine the modification sites.

[0009] This invention provides a method for detecting protein PEGylation modification sites, the method comprising the following steps:

[0010] (1) Enzymatic digestion of PEGylated proteins;

[0011] (2) The polypeptide obtained after enzymatic digestion in step (1) is separated into PEG-related peptides by liquid chromatography-mass spectrometry, and the collected (peptide first-level) mass spectrometry data is analyzed to determine the PEG-related peptides.

[0012] (3) Break up all the primary mass spectrometry peaks obtained in step (2) by secondary mass spectrometry, use any other PEG-related peak as background, subtract the background from the peak to be analyzed, and obtain the measured values ​​of b and y ions.

[0013] (4) Input any adjacent peptide sequences generated after enzyme digestion in step (1) into software (e.g., the MS-Digest module of ProteinProspector) to obtain the theoretical values ​​of b and y ions. Match the measured values ​​of b and y ions obtained in step (3) with the theoretical values ​​to determine the modification sites.

[0014] In particular, step (3) involves breaking down all the primary mass spectrometry peaks obtained in step (2) using secondary mass spectrometry without performing parent ion screening.

[0015] Furthermore, the secondary mass spectrometry data acquisition method described in step (3) is a data information-dependent acquisition mode (such as IDA) or an information-independent acquisition mode (such as Swath).

[0016] Preferably, the secondary mass spectrometry data acquisition adopts an information-independent acquisition mode.

[0017] The protein may be selected from: interleukin (IL), antibody, antibody-drug conjugate, coagulation factor, etc.; the molecular weight of the PEG is 1×10⁻⁶. 4 Da~5×10 4 Da (such as 1×10 4 Da, 1.1×10 4 Da, 1.2×10 4 Da, 1.3×10 4 Da, 1.4×10 4 Da, 1.5×10 4 Da, 1.6×10 4 Da, 1.7×10 4 Da, 1.8×10 4 Da, 1.9×10 4 Da, 2×10 4 Da, 2.1×10 4 Da, 2.2×10 4 Da, 2.3×10 4 Da, 2.4×10 4 Da, 2.5×10 4 Da, 2.6×10 4 Da, 2.7×10 4 Da, 2.8×10 4 Da, 2.9×10 4 Da, 3×10 4 Da, 3.5×10 4Da, 4×10 4 Da, 4.5×10 4 Da, 5×10 4 (da), preferably, the molecular weight of the PEG is 1×10. 4 Da~4×10 4 More preferably, the molecular weight of the PEG is 1×10⁻⁶. 4 Da~3×10 4 Da.

[0018] In one embodiment of the present invention, the molecular weight of the PEG is 20 kDa.

[0019] In one embodiment of the present invention, the protein is an interleukin (IL).

[0020] In one embodiment of the invention, the protein is interleukin (IL-2).

[0021] Furthermore, step (1) includes diluting, reducing, alkylating and changing the solution of the PEGylated protein before enzymatic digestion of the PEGylated protein.

[0022] Further, the concentration of the dilution is 0.05-0.5 mg / ml (e.g., 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml, 0.3 mg / ml, 0.35 mg / ml, 0.4 mg / ml, 0.45 mg / ml, 0.5 mg / ml), preferably 0.05-0.4 mg / ml, more preferably 0.05-0.3 mg / ml, and even more preferably 0.1-0.3 mg / ml.

[0023] In one embodiment of the present invention, the concentration of the dilution is 0.25 mg / ml.

[0024] Furthermore, the reduction includes the step of mixing a reducing agent with a PEGylated protein.

[0025] Further, the mixing time between the reducing agent and the PEGylated protein is 0.3 to 5 hours (e.g., 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 hours), preferably 0.3 to 3 hours, and more preferably 0.3 to 1 hour.

[0026] In one embodiment of the present invention, the reducing agent is mixed with the PEGylated protein for 0.5 h.

[0027] Furthermore, the temperature at which the reducing agent is mixed with the PEGylated protein is 25–40°C (e.g., 25°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C), preferably 30–40°C.

[0028] In one embodiment of the present invention, the reducing agent is mixed with the PEGylated protein at a temperature of 37°C.

[0029] Furthermore, the reducing agent used in the reduction step can be dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), preferably DTT.

[0030] Further, the final concentration of the reducing agent is 5–100 mmol / L (e.g., 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L), preferably 5–50 mmol / L, and more preferably 5–20 mmol / L.

[0031] In one embodiment of the present invention, the final concentration of the reducing agent is 10 mmol / L.

[0032] Furthermore, the alkylation includes the step of mixing the alkylating agent with the aforementioned reduced PEGylated protein.

[0033] Furthermore, the concentration of the alkylating agent is 2-4 times the concentration of the reducing agent, preferably 2.5-4 times the concentration of the reducing agent.

[0034] In one embodiment of the present invention, the concentration of the alkylating agent is twice that of the reducing agent.

[0035] Furthermore, the alkylating agent used in the alkylation step can be iodoacetamide (IAM).

[0036] Furthermore, the fluid exchange includes the steps of mixing the aforementioned alkylated PEGylated protein with ammonium bicarbonate and ultrafiltration.

[0037] Furthermore, the rotation speed of the ultrafiltration step is 8000-18000 rpm, preferably 10000-15000 rpm.

[0038] In one embodiment of the present invention, the rotation speed of the ultrafiltration step is 12,000 rpm.

[0039] Furthermore, the ultrafiltration step takes 5 to 25 minutes, preferably 10 to 20 minutes.

[0040] Further, the temperature of the ultrafiltration step is 4 to 25°C (e.g., 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 20°C, 25°C), preferably 8 to 12°C.

[0041] In one embodiment of the present invention, the temperature of the ultrafiltration step is 10°C.

[0042] Furthermore, the ultrafiltration step is performed 1 to 5 times, preferably 2 to 4 times.

[0043] In one embodiment of the present invention, the ultrafiltration step is performed three times.

[0044] Further, the concentration of the PEGylated protein after ultrafiltration is 0.05–0.5 mg / ml (e.g., 0.05 mg / ml, 0.1 mg / ml, 0.11 mg / ml, 0.12 mg / ml, 0.13 mg / ml, 0.14 mg / ml, 0.15 mg / ml, 0.16 mg / ml, 0.17 mg / ml, 0.18 mg / ml, 0.19 mg / ml, 0.2 mg / ml, 0.21 mg / ml, 0.22 mg / ml, 0.23 mg / ml, 0.24 mg / ml, 0.25 mg / ml, 0.3 mg / ml, 0.35 mg / ml, 0.4 mg / ml, 0.45 mg / ml, 0.5 mg / ml), preferably 0.05–0.3 mg / ml, more preferably 0.1–0.3 mg / ml.

[0045] In one embodiment of the present invention, the concentration of the PEGylated protein after ultrafiltration is 0.17 mg / ml.

[0046] Further, the enzymatic digestion described in step (1) includes the step of mixing the PEGylated protein with an enzyme.

[0047] Further, the ratio of the PEGylated protein to the enzyme is 10–30:0.5–5 (e.g., 10–30:0.5–5, 15–25:0.5–2, 10–30:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 20: 0.1~5, 20:0.1, 20:0.5, 20:0.6, 20:0.7, 20:0.8, 20:0.9, 20:1, 20:1.1, 20:1.2, 20:1.3, 20:1.4, 20:1.5, 20:1.6, 20:1.7, 20:1.8, 20:1.9, 20:2, 20:2.5, 20:3, 20:3.5, 20:4, 20:4.5, 20:5), preferably 15~25:0.5~2.

[0048] In one embodiment of the present invention, the ratio of the PEGylated protein to the enzyme is 20:1.

[0049] Furthermore, the enzyme is a specific protein endonuclease, which is selected from one or more combinations of trypsin, chymotrypsin, lysine C-terminal protease, arginine C-terminal protease and Staphylococcus aureus V-8 protease.

[0050] Preferably, the specific protein endonuclease is selected from one or more combinations of trypsin, lysine C-terminal protease, and arginine C-terminal protease.

[0051] In one embodiment of the present invention, the specific protein endonuclease is trypsin.

[0052] Furthermore, the temperature at which the PEGylated protein is mixed with the enzyme is 30°C to 40°C (e.g., 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C).

[0053] In one embodiment of the invention, the mixing temperature is 37°C.

[0054] Furthermore, the mixing time between the PEGylated protein and the enzyme is 2 to 20 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours).

[0055] In one embodiment of the present invention, the mixing time is 12 hours.

[0056] Furthermore, step (1) after enzymatically digesting the PEGylated protein also includes a step to terminate the reaction.

[0057] Furthermore, the termination reaction includes the step of adding formic acid solution to the enzymatically digested PEGylated protein.

[0058] Further, the final mass concentration of the formic acid solution is 0.5% to 5% (e.g., 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%).

[0059] In one embodiment of the present invention, the final mass concentration of the formic acid solution is 1%.

[0060] Furthermore, the liquid chromatography described in step (2) is reversed-phase liquid chromatography.

[0061] Further, the mobile phase of the liquid chromatography in step (2) includes mobile phase A and mobile phase B, where mobile phase A is an aqueous solution of formic acid and mobile phase B is an acetonitrile solution of formic acid, and mobile phase A + mobile phase B = 100%.

[0062] Further, the mobile phase A is a 0.01-0.5% formic acid aqueous solution, preferably, the mobile phase A is a 0.1% formic acid aqueous solution.

[0063] Further, the mobile phase B is a 0.01-0.5% formic acid acetonitrile solution, preferably, the mobile phase B is a 0.1% formic acid acetonitrile solution.

[0064] Further, the flow rate of the mobile phase is 0.05–1 mL / min (e.g., 0.05 mL / min, 0.06 mL / min, 0.07 mL / min, 0.08 mL / min, 0.09 mL / min, 0.1 mL / min, 0.11 mL / min, 0.12 mL / min, 0.13 mL / min, 0.14 mL / min, 0.15 mL / min, 0.16 mL / min, 0.17 mL / min, 0.18 mL / min, 0.19 mL / min, 0.2 mL / min), preferably, the flow rate of the mobile phase is 0.1–0.5 mL / min.

[0065] In one embodiment of the present invention, the flow rate of the mobile phase is 0.2 mL / min.

[0066] Further, the column temperature of the chromatographic column of the liquid chromatography is 30-70℃ (e.g., 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 45℃, 50℃, 60℃, 70℃), preferably, the column temperature of the chromatographic column is 35-45℃.

[0067] In one embodiment of the present invention, the column temperature of the chromatographic column is 40°C.

[0068] Further, the injection volume of the liquid chromatograph is 1 to 15 μL (e.g., 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 15 μL), preferably, the injection volume of the liquid chromatograph is 2 to 12 μL.

[0069] In one embodiment of the present invention, the injection volume of the liquid chromatograph is 5 μL.

[0070] In one embodiment of the present invention, the injection volume of the liquid chromatograph is 10 μL.

[0071] Furthermore, the liquid chromatography employs a gradient elution program.

[0072] In one embodiment of the present invention, the gradient elution procedure is as follows:

[0073] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 0.2 98 2 4 0.2 98 2 24 0.2 84 16 34 0.2 74 26 64 0.2 60 40 100.5 0.2 10 90 102.5 0.2 10 90 103 0.2 98 2 108 0.2 98 2

[0074] In one embodiment of the present invention, the gradient elution procedure is as follows:

[0075] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 0.2 70 30 2 0.2 65 35 30 0.2 45 55 31 0.2 10 90 33 0.2 10 90 34 0.2 70 30 40 0.2 70 30

[0076] In one embodiment of the present invention, the mass spectrometry parameters are as follows:

[0077]

[0078]

[0079] In one embodiment of the present invention, the mass spectrometry parameters are as follows:

[0080]

[0081] The second aspect of this invention provides the application of the above-described detection method in the identification of protein PEGylation modification sites.

[0082] This invention involves cleaving PEG-modified proteases with large molecular weights into peptides. Instead of screening for precursor ions, the PEGylated peptides are directly fragmented into secondary fragments, selecting the portion (IDA mode) or all (SWATH mode) with the highest primary signal. Interference from PEG fragments is eliminated through background subtraction, specifically targeting PEG. The secondary signal of the PEGylated peptides is directly analyzed. When the modification site is lysine, trypsin or Lys-C enzyme cannot cleave the modified lysine. Utilizing this characteristic, all hypothetical peptide sequences formed after PEGylation can be listed, rather than directly comparing the fragments after enzymatic digestion to a database to obtain theoretical b and y ions. By comparing each actual generated ion, the corresponding modified peptide can be matched more accurately, resulting in more reliable identification results. Attached Figure Description

[0083] Figure 1 This is a TIC plot of the IL2-PEG peptide map in IDA mode.

[0084] Figure 2 This is a magnified view of the PEG-related peptide segments in the TIC diagram of the IL2-PEG peptide map under IDA mode.

[0085] Figure 3 The second-order mass spectra of PEG-related peaks in IDA mode (where A is...) Figure 2 Figure B shows the secondary mass spectrum of peak 1 after background subtraction; Figure B is... Figure 2 The secondary mass spectrum of peak 2 after background subtraction; Figure C is... Figure 2 The secondary mass spectrum of peak 3 after background subtraction; Figures D and E are... Figure 2 (Secondary mass spectrum of peak 4 after background subtraction).

[0086] Figure 4 TIC plot of IL2-PEG peptide in Swath mode.

[0087] Figure 5 The following are secondary mass spectra of PEG-related peaks in Swath mode (where A and B are...). Figure 4 The secondary mass spectrum of peak 1 after background subtraction; Figure C is... Figure 4 The secondary mass spectrum of peak 2 after background subtraction; Figure D is... Figure 4 The secondary mass spectrum of peak 3 after background subtraction; Figures E and F are... Figure 4 (Secondary mass spectrum of peak 4 after background subtraction). Detailed Implementation

[0088] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0089] Example 1: IDA fragmentation mode was used to determine PEG modification sites.

[0090] The modification sites of 20 kDa PEG-modified IL2 were analyzed. The IL2 was crosslinked with an average of one PEG molecule and was prepared by Keyk Technology Co., Ltd. The sequence can be viewed by entering "interleukin 2" and selecting Homo sapiens on the NCBI website.

[0091] The specific method is as follows:

[0092] 1. Instrument parameters

[0093] Instrument: SCIEX TripleTOF6600

[0094] Liquid phase parameters

[0095]

[0096]

[0097] Liquid phase gradient

[0098] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 0.2 98 2 4 0.2 98 2 24 0.2 84 16 34 0.2 74 26 64 0.2 60 40 100.5 0.2 10 90 102.5 0.2 10 90 103 0.2 98 2 108 0.2 98 2

[0099] Mass spectrometry parameters

[0100]

[0101] 2. Sample and solution preparation

[0102] 2.1 Reagent Preparation

[0103] (1) 50mM ammonium bicarbonate solution: Weigh 0.198g of ammonium bicarbonate into a 50ml centrifuge tube, add ultrapure water to the 50ml mark, vortex to dissolve, and store at 4℃.

[0104] (2) Preparation of 1M DTT solution: Weigh 0.150g of DTT into a 1.5ml centrifuge tube, add 1ml of ultrapure water, mix well, dispense into 20μl / tube, and store at -20℃.

[0105] (3) Preparation of 0.5M IAM solution: Weigh 0.092g of iodoacetamide into a 1.5ml centrifuge tube and add 1ml of ultrapure water. Mix well, dispense into 50μl tubes, and store at -20℃.

[0106] 2.2 Sample Pretreatment

[0107] (1) Sample dilution: Dilute the sample with 50mM ammonium bicarbonate to 0.25mg / ml, with a final volume of 100μl.

[0108] (2) Reduction: Take the diluted sample, add 1 μl of 1M DTT, and react at 37℃ for 0.5 h.

[0109] (3) Alkylation: Add 4 μl of 0.5 mol / L IAM to make the final concentration about 20 mmol / L, mix well, and let stand at room temperature in the dark for 45 min.

[0110] (4) Solution replacement: Add 400 μl of 50 mM ammonium bicarbonate to the above sample, transfer to a 3K ultrafiltration tube, and ultrafilter at 12000 rpm and 10℃ for 15 min. When approximately 100 μl of sample remains, add another 400 μl of 50 mM ammonium bicarbonate and continue ultrafiltration. Repeat this process 3 times until the remaining volume is approximately 120 μl. The concentration is approximately 0.17 mg / ml.

[0111] (5) Enzymatic hydrolysis: Take 120 μl of the above solution and add 10 μl of 0.1 mg / ml trypsin at a protein:enzyme ratio of 20:1. React overnight at 37°C.

[0112] (6) Termination of reaction: Take 180 μl of ultrapure water and add 20 μl of formic acid to prepare 10% formic acid solution. Prepare fresh for each use. Add 6 μl of the 10% formic acid solution to the enzyme-digested sample to terminate the reaction. Transfer the sample to a vial and detect by LC-MS.

[0113] 3. Results Analysis

[0114] Because 20K PEG is a polymer with a wide molecular weight range, the signal could not be determined by deconvolution in primary mass spectrometry. Therefore, the modified peptides were identified by manually analyzing its secondary mass spectra.

[0115] The cleavage site of trypsin is the C-terminus of lysine (K) and arginine (R) (it cannot be cleaved when the last amino acid is proline). When PEG is cross-linked with the K side chain of IL2, trypsin cannot cleave it. The modification site can be determined by the b and y ions generated by the fragmentation of two adjacent peptides via CID. Since PEG fragmentation generates a large number of interfering ions, background subtraction is required.

[0116] Peak 4 (reference) Figure 2 Taking (number) as an example, the main process of result analysis is as follows:

[0117] ① Determine PEG peptide peaks: Based on the liquid phase retention time delay after cross-linking PEG and PEG characteristic fragment ions such as 133 and 177, determine PEG-related peptides.

[0118] ② Background subtraction: Open the secondary mass spectra of peak 1 and peak 4, and subtract the PEG-related ions in peak 4 using peak 1 as the background.

[0119] ③ List the theoretical b and y ions: Since trypsin cannot cleave the peptide at K after cross-linking PEG, a missed cleavage peptide will be generated. List the theoretical b and y ions for all 1 missed cleavage peptides. The theoretical ions can be predicted by MS-digest on the ProteinProspector website.

[0120] ④ Comparison between measured and theoretical values: Compare the ions with higher abundance in the secondary mass spectrum after background subtraction in ① with the theoretical b and y ions. If they can match a certain peptide segment with a large number of b and y ions, then the PEG modification is considered to have occurred in that peptide segment.

[0121] (1) TIC plot analysis of IL2-PEG

[0122] PEG cross-linked peptides can be separated by liquid chromatography. IL2-1-PEG has 4 separation peaks with an elution window of 70-80 min, indicating that there are mainly 4 cross-linking sites.

[0123] See details Figure 1 and Figure 2 .

[0124] (2) Secondary mass spectrometry to determine modification sites

[0125] Based on the above analytical methods, the secondary b and y ion diagrams related to its peptides were obtained (e.g., Figure 3 (As shown), and then compared one by one with the theoretical sequence of IL2 after trypsin digestion containing one missed cleavage, to determine the modification sites corresponding to the four peaks. The results are as follows (modified amino acids and matching b and y ions are underlined):

[0126] Peak 1: HLQCLEEELKPLEEVLNLAQS K NFHLRPR

[0127] List of peptide theory b and y ions (underlined ions are matching daughter ions)

[0128]

[0129]

[0130] Peak 2: K ATELK

[0131] List of peptide theory b and y ions (underlined ions are matching daughter ions)

[0132] b-series ions Residue molecular weight amino acids Y-series ions Residue molecular weight b1 --- K y6 --- b2 200.1394 A y5 <![CDATA[ 561.3243 ]]> b3 301.187 T y4 <![CDATA[ 490.2871 ]]> b4 430.2296 E y3 <![CDATA[ 389.2395 ]]> b5 543.3137 L y2 <![CDATA[ 260.1969 ]]> b6 --- K y1 147.1128

[0133] Peak 3: A PTSSSTK

[0134] List of peptide theory b and y ions (underlined ions are matching daughter ions)

[0135] b-series ions Residue molecular weight amino acids Y-series ions Residue molecular weight b1 --- A y8 --- b2 169.0972 P y7 <![CDATA[ 707.357 ]]> b3 270.1448 T y6 <![CDATA[ 610.3042 ]]> b4 357.1769 S y5 <![CDATA[ 509.2566 ]]> b5 444.2089 S y4 <![CDATA[ 422.2245 ]]> b6 531.2409 S y3 <![CDATA[ 335.1925 ]]> b7 632.2886 T y2 <![CDATA[ 248.1605 ]]> b8 --- K y1 147.1128

[0136] Peak 4: TQLQLEHLLLDLQMILNGINNY K NPK

[0137] List of peptide theory b and y ions (underlined ions are matching daughter ions)

[0138]

[0139]

[0140] Example 2 uses the Swath fragmentation model to determine PEG modification sites.

[0141] 1. Instrument Information

[0142] Instrument: SCIEX TripleTOF6600

[0143] Liquid phase parameters

[0144]

[0145] Liquid phase gradient

[0146] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 0.2 70 30 2 0.2 65 35 30 0.2 45 55 31 0.2 10 90 33 0.2 10 90 34 0.2 70 30 40 0.2 70 30

[0147] Mass spectrometry parameters

[0148]

[0149]

[0150] 2. Sample and solution preparation

[0151] Same as Example 1.

[0152] 3. Results Analysis

[0153] The PEG-crosslinked peptides were separated by liquid chromatography. IL2-1-PEG showed four separation peaks. After optimizing the liquid chromatography gradient, the TIC chromatogram peak window was 14-22 min, mainly showing four crosslinking sites. See details... Figure 4 .

[0154] The secondary mass spectrum of the PEG-related peaks in Swath mode is shown below. Figure 5 .

[0155] Peak 1: HLQCLEEELKPLEEVLNLAQS K NFHLRPR

[0156] List of peptide theory b and y ions (underlined ions are matching daughter ions)

[0157]

[0158]

[0159] Peak 2: K ATELK

[0160] List of peptide theory b and y ions (underlined ions are matching daughter ions)

[0161] b-series ions Residue molecular weight amino acids Y-series ions Residue molecular weight b1 --- K y6 --- b2 200.1394 A y5 561.3243 b3 301.187 T y4 <![CDATA[ 490.2871 ]]> b4 430.2296 E y3 <![CDATA[ 389.2395 ]]> b5 543.3137 L y2 <![CDATA[ 260.1969 ]]> b6 --- K y1 147.1128

[0162] Peak 3: A PTSSSTK

[0163] List of peptide theory b and y ions (underlined ions are matching daughter ions)

[0164] b-series ions Residue molecular weight amino acids Y-series ions Residue molecular weight b1 --- A y8 --- b2 169.0972 P y7 <![CDATA[ 707.357 ]]> b3 270.1448 T y6 <![CDATA[ 610.3042 ]]> b4 357.1769 S y5 <![CDATA[ 509.2566 ]]> b5 444.2089 S y4 <![CDATA[ 422.2245 ]]> b6 531.2409 S y3 <![CDATA[ 335.1925 ]]> b7 632.2886 T y2 <![CDATA[ 248.1605 ]]> b8 --- K y1 147.1128

[0165] Peak 4: TQLQLEHLLLDLQMILNGINNY K NPK

[0166] List of peptide theory b and y ions (underlined ions are matching daughter ions)

[0167]

[0168]

[0169] The response value of the second-order mass spectrometry was improved from <10 in IDA mode to several hundred.

[0170] The sites determined by both methods are the same, as shown in the table below:

[0171] Peak site Peak 1 <![CDATA[HLQCLEEELKPLEEVLNLAQS K NFHLRPR]]> Peak 2 <![CDATA[ K ATELK]]> Peak 3 <![CDATA[ A PTSSSTK]]> Peak 4 <![CDATA[TQLQLEHLLLDLQMILNGINNY K NPK]]>

Claims

1. A method for detecting protein PEGylation modification sites, the method comprising the following steps: (1) The PEGylated protein was digested with enzymes; the molecular weight of the PEG was 1×10⁻⁶. 4 Da~5×10 4 Da; (2) The polypeptide obtained after enzymatic digestion in step (1) is separated into PEG-related peptides by liquid chromatography-mass spectrometry, and the collected mass spectrometry data is analyzed to determine the PEG-related peptides; the enzyme is trypsin. (3) Without screening the precursor ion, the primary mass spectrum peak obtained in step (2) is broken up by secondary mass spectrometry. Any other PEG-related peak is used as the background. The secondary mass spectrum of the peak to be analyzed is subtracted from the background to obtain the measured values ​​of b and y ions. The secondary mass spectrometry data acquisition method is either a data information-dependent acquisition mode or an information-independent acquisition mode. (4) Input any adjacent peptide sequence generated after enzyme digestion in step (1) into the software. Since the trypsin cannot cut the peptide at K after cross-linking PEG, a missed peptide will be generated. List the theoretical b and y ions of all 1 missed peptides, obtain the theoretical values ​​of b and y ions, and match the measured values ​​of b and y ions obtained in step (3) with the theoretical values ​​to determine the modification site.

2. The detection method as described in claim 1, characterized in that, The ratio of the PEGylated protein to the enzyme is 10~30:0.5~5.

3. The detection method as described in claim 1, characterized in that, The ratio of the PEGylated protein to the enzyme is 15~25:0.5~2.

4. The detection method as described in claim 1, characterized in that, The ratio of the PEGylated protein to the enzyme was 20:

1.

5. The detection method as described in claim 1, characterized in that, The liquid chromatography described in step (2) is reversed-phase liquid chromatography; The mobile phase of the liquid chromatography in step (2) includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid, and mobile phase B is an aqueous solution of formic acid and acetonitrile. The mobile phase A + mobile phase B = 100%. The flow rate of the mobile phase is 0.05~1 mL / min.

6. The detection method as described in claim 5, characterized in that, The mobile phase A is a 0.01~0.5% formic acid aqueous solution.

7. The detection method as described in claim 6, characterized in that, The mobile phase A is a 0.1% formic acid aqueous solution.

8. The detection method as described in claim 5, characterized in that, The mobile phase B is a 0.01~0.5% formic acid acetonitrile solution.

9. The detection method as described in claim 8, characterized in that, The mobile phase B is a 0.1% formic acid acetonitrile solution.

10. The detection method as described in claim 5, characterized in that, The protein may be selected from: interleukins, antibodies, antibody-drug conjugates, and coagulation factors.

11. The detection method as described in claim 10, characterized in that, The protein in question is interleukin.

12. The detection method as described in claim 11, characterized in that, The protein in question is interleukin.

13. The detection method as described in claim 5, characterized in that, The molecular weight of the PEG is 1×10⁻⁶. 4 Da~4×10 4 Da.

14. The detection method as described in claim 13, characterized in that, The molecular weight of the PEG is 1×10⁻⁶. 4 Da~3×10 4 Da.

15. The application of the detection method as described in any one of claims 1-14 in the identification of protein PEGylation modification sites.

Images