Method for improving protein methylation modification identification analysis
The hydrophobic peptide peptide is increased through hydrophobic derivation modification technology, which solves the problem of weak retaining in the anti -phase chromatography, and significantly improves the appraisal efficiency and coverage of methylized peptides.
Patent Information
- Application Number
- CN202310922408.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-26
- Publication Date
- 2025-01-28
AI Technical Summary
Protein methylated peptides are weakly retained in reverse phase chromatography, resulting in low mass spectrometry identification efficiency, especially short-chain methylated peptides.
Through hydrophobic derivation modification technology, the hydrophobicity of methylized peptides is increased, thereby extending its retention time in the anti -phase chromatography and improving the appraisal efficiency.
It significantly improves the appraisal efficiency of protein methylized peptides, especially the appraisal coverage of short -chain methyl -based peptides, and enhances the appraisal coverage of the methylized protein group.
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Figure CN119375485A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the research field of protein post-translational modification in the research direction of proteomics, and specifically relates to a method for improving protein methylation identification based on hydrophobic derivatization modification. Background Art
[0002] Protein methylation is one of the important post-translational modifications of proteins and plays a key regulatory role in various biological processes. Methylation modification does not change the physical and chemical properties of peptides, so it is very difficult to effectively identify and analyze protein methylation modification. In order to overcome this scientific challenge, a large number of methods have been developed to identify protein methylation (Reference 1: Ma, M.; Zhao, XY; Chen, S.; Zhao, YY; Yang, L.; Feng, Y.; Qin, WJ; Li, LJ; Jia, C-X. Strategy based on deglycosylation, multiprotease, and hydrophilic interaction chromatography for large-scale profiling of protein methylation. Analytical Chemistry, 2017, 89(23), 12909-12917; Reference 2: Wu, ZX; Cheng, ZY; Sun, MW; Wan, XL; Liu, P.; He, TM; Tan, MJ; Zhao, YM A. Chemical proteomics approach for global analysis of lysine monomethylome profiling. Molecular & Cellular Proteomics, 2015, 14(2), 329-339; Document 3: Guo, AL; Gu, HB; Zhou, J.; Mulhern, D.; Wang, Y.; Lee, KA; Yang, V.; Aguiar, M.; Kornhauser, J.; Jia, Proteomics,2014,13(1),372-387).However, methylated peptides have strong hydrophilicity (Reference 1: Ma, M.; Zhao, XY; Chen, S.; Zhao, YY; Yang, L.; Feng, Y.; Qin, WJ; Li, LJ; Jia, CXStrategy based on deglycosylation, multiprotease, and hydrophilic interaction chromatography for large-scale profiling of proteinmethylation. Analytical Chemistry, 2017, 89(23), 12909-12917), and are difficult to be effectively retained in reversed-phase chromatography, resulting in low efficiency of mass spectrometry for methylated peptide identification, especially for short-chain methylated peptides. Therefore, enhancing the hydrophobicity of methylated peptides is crucial to improve the identification coverage of methylated proteomes.
[0003] In order to improve the identification efficiency of protein methylation peptides, the present invention proposes a method for improving protein methylation identification based on hydrophobic derivatization modification. Summary of the invention
[0004] The purpose of the present invention is to provide a method which is simple, low-cost, and highly reproducible and can significantly improve the identification of protein methylation modification.
[0005] A method for improving protein methylation identification based on hydrophobic derivatization modification. The method is based on the fact that methylated peptides are weakly retained in reversed-phase chromatography, resulting in low identification efficiency by mass spectrometry. Hydrophobic derivatization will increase the retention time of methylated peptides in reversed-phase chromatography and improve the identification efficiency of methylated peptides.
[0006] The specific steps of the method for improving protein methylation modification identification and analysis are as follows:
[0007] (1) enzymatically hydrolyzing a protein solution of a biological sample into peptide fragments to obtain a protease hydrolysate;
[0008] The peptides in the obtained protease hydrolysate are subjected to hydrophobic derivatization modification by using a hydrophobic derivatization strategy, and the protease hydrolysate in which the peptides are subjected to hydrophobic derivatization modification serves as a derivatization modification group;
[0009] The protease digest of the peptides without derivatization modification was used as the control group;
[0010] (2) Enriching the methylated peptides in the protease hydrolysates of the control group and the derivatization modification group respectively;
[0011] (3) Using mass spectrometry to analyze the methylated peptides in the control group and the derivatization modification group;
[0012] (4) Using search software to search the files generated by mass spectrometry, the methylation sites of the control group and the derivatization modification group were obtained respectively;
[0013] By combining the methylation sites identified in the control group and the derivatization modification group (i.e., adding the number of methylation sites in the control group and the derivatization modification group and then deleting the number of repeated methylation sites, i.e., taking 1 as the number of two repeated sites), the site information of protein methylation in the biological sample can be obtained.
[0014] The protein solution of the biological sample includes one or more of cell and / or tissue extracts; the cell and / or tissue extracts are derived from one or more of cells and / or tissues of animals, plants, cells or humans; the biological sample protein solution is obtained by lysing the tissue and / or cells with a buffer solution, so that the protein solution in the tissue and / or cells is denatured; the preferred buffer solution contains 1-10M urea, 10-100mM dithiothreitol and 5-100mM tris(hydroxymethyl)aminomethane solution with a volume concentration of 0.5-2% polyethylene glycol octylphenyl ether, and the tissue and / or cells are ultrasonically broken and lysed in the buffer solution; the preferred protein solution of the tissue and / or cells is obtained by metabolically labeling L-methionine-(methyl-CH 3 ) into L-methionine-(methyl- 13 C,D 3 ), which helps to improve the credibility of protein methylation identification.
[0015] The proteolysis process is carried out in two steps. In the first step, the proteases used are any two proteases with different cleavage sites among trypsin, lysinase Lys-C, arginase Arg-C, Glu-C protease, pepsin, etc., but are not limited to these proteases; the usage ratio of the two proteases with different cleavage sites to the protein is 1:10-1:100 (the mass ratio of the protease to the protein to be hydrolyzed); the protein solution of the tissue and / or cell is subjected to the first enzymatic hydrolysis using two proteases with different cleavage sites (the mass ratio of the protease to the protein to be hydrolyzed is 1:10-1:100, preferably 1:50), such as Glu-C protease (specifically hydrolyzes the carboxyl terminal peptide bond of glutamic acid and aspartic acid) and pepsin (specifically hydrolyzes aromatic amino acids, such as phenylalanine, tyrosine, leucine and tryptophan). The first step is to use a protease with a different cleavage site from the first two proteases (the ratio of the protease to the protein to be hydrolyzed is 1:10-1:100, preferably 1:50) to further hydrolyze the protease with two cleavage sites, such as lysinase Lys-C (specifically hydrolyzes the carboxyl-terminal peptide bond of lysine), to obtain a protease with three cleavages; through two-step protease hydrolysis, a shorter enzymatic peptide segment is obtained, which is conducive to hydrophobic derivatization modification (the peptide segment is longer, and the hydrophobicity of the peptide segment will become stronger after hydrophobic derivatization modification, and it has a stronger retention in reverse phase chromatography and is difficult to be eluted, resulting in a decrease in the identification efficiency of the methylated peptide segment); through two-step protease hydrolysis, the missed cleavage rate of the peptide segment is effectively reduced, which is conducive to the subsequent separation and enrichment of the methylated peptide segment.
[0016] The hydrophobic derivatized group may be one or more of the thiol group of cysteine, the carboxyl group of the peptide, the amino group of the peptide, etc., but are not limited to these groups; the thiol group of cysteine may be one or more of N,N-dimethyl-2-chloroethylamine, iodoacetanilide, N-ethylmaleimide, etc., but are not limited to these reaction reagents; the carboxyl group may be one or more of 1-(2-pyrimidine)piperazine, 2-p-nitrophenylhydrazine, benzylamine, 1-benzylpiperazine, etc., but are not limited to these reaction reagents; the amino group may be one or more of coumarin derivatives, sulfonyl chloride, alkyl aldehyde, diisopropyl phosphite, etc., but are not limited to these reaction reagents.
[0017] The preferred derivatization reagent is an alkyl aldehyde that modifies the amino group, and the alkyl aldehyde includes one or more of acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, hexanal, etc., but is not limited to these aldehydes; the derivatization process is as follows: using a buffer containing 50-200mM alkyl aldehyde and 50-200mM pyridine and an enzymatic peptide fragment with a final concentration of 0.1-10μg / μl to carry out a first reaction in a 20-30°C water bath for 1-6h; then adding a buffer containing 100-500mM alkyl aldehyde and 100-500mM pyridine to the first reaction solution for 20-30 The second reaction is carried out in a 50°C water bath for 1-24 hours; the volume ratio of the buffer added for the second time to the solution of the first reaction is 0.5:1-3:1; due to the steric hindrance of the aldehyde group, the N-terminus of most peptides and the primary amine group of lysine in the first derivatization reaction only react with one aldehyde group; in order to make the N-terminus of each peptide and the primary amine group of lysine react with two aldehyde groups, a second derivatization reaction is carried out to enhance the hydrophobicity of the peptides, which is conducive to improving the identification of methylated peptides; the preferred buffer is 10-100mM 4-hydroxyethylpiperazineethanesulfonic acid buffer.
[0018] The obtained peptides are hydrophobically derivatized using a hydrophobic derivatization strategy to serve as a derivatization modification group; the peptides that are not derivatized are used as a control group; the methylated peptides in the control group and the derivatization modification group are enriched using chromatography, antibodies, recognition domains and other methods, but are not limited to the above methods; the chromatographic method includes one or more of affinity chromatography, strong cation exchange chromatography, strong anion exchange chromatography, etc., but is not limited to these chromatographic methods; the antibody method includes one or more of lysine methylation antibodies, arginine methylation antibodies, etc., but is not limited to these antibodies; the recognition domains include MBT domains (malignant brain tumor domains), Tudor domains, PHD domains (plant homeodomain fingers), chromatin domains (chromodomains), PWWP domains (proline-tryptophan-tryptophan-proline domains), WD40 domains (WD40 repeat domains), BAH domains (bromo adjacent homology domains) and ANK domains (ankyrin repeats) and one or more of them, but not limited to these domains; the preferred method for separating and enriching the methylated peptides of the control group and the derivatization modification group is strong cation exchange chromatography, including one or more of cation exchange groups such as methyl sulfonate and carboxylmethyl, but not limited to these exchange groups; the separation and enrichment process is as follows: 1-200 μL of strong cation exchange chromatography material is added to tips (solid phase extraction pipette tip / gun tip, using the absorbent cotton loaded in the tip of the gun tip as a sieve plate), respectively, and washed 1-3 times with 100-1000 μL of washing buffer I, and then washed 1-3 times with 100-1000 μL of washing buffer II, and equilibrated 1-3 times with 100-1000 μL of equilibration buffer, respectively, and the obtained control group and derivatization modification group are separated and enriched. The enzymatic peptides of the derivatization modification group were respectively redissolved in 100-1000 μL of equilibrium buffer, and added to the tips (solid phase extraction pipette tip / gun tip, using the absorbent cotton loaded in the tip of the gun tip as a sieve plate) containing strong cation exchange chromatography materials, and the enzymatic peptides of the derivatization modification group were washed 1-3 times with 100-1000 μL of washing buffer III, and the enzymatic peptides of the control group were washed 1-3 times with 100-1000 μL of washing buffer IV, and eluted 1-3 times with 100-1000 μL of elution buffer, respectively, and the obtained peptide eluates were freeze-dried to obtain methylated peptides of the control group and the derivatization modification group; the washing buffer I was 1-10 mM Bo-Rodriguez (1-10 mM acetic acid, 1-10 mM phosphoric acid and 1-10 mM boric acid), and the pH value was 10-14;Wash buffer II is 1-10 mM Br-Rodriguez (1-10 mM acetic acid, 1-10 mM phosphoric acid and 1-10 mM boric acid), pH 1-5; balance buffer is 10-60% acetonitrile, 40-90% 1-10 mM Br-Rodriguez (1-10 mM acetic acid, 1-10 mM phosphoric acid and 1-10 mM boric acid), pH 1-5; wash buffer III is 80-100% acetonitrile, 0-20% 1-10 mM Br-Rodriguez The pH value is 1-10; the washing buffer IV is 70-100% acetonitrile, 0-30% 1-10mM Brom-Rodriguez (1-10mM acetic acid, 1-10mM phosphoric acid and 1-10mM boric acid), and the pH value is 1-10; the elution buffer is a mixed solution of 10-60% acetonitrile and 40-90% ammonia water with a mass concentration of 1-50%.
[0019] The methylated peptides of the control group and the derivatization modification group were respectively redissolved in trifluoroacetic acid with a volume concentration of 0.05-1% for RP LC-MS / MS analysis.
[0020] The obtained mass spectrum files are searched respectively using one or more software such as MaxQuant, pFind, etc., but the search software is not limited to these.
[0021] The method is based on the fact that the weak retention of methylated peptides in reversed-phase chromatography leads to low identification efficiency of methylated peptides, and hydrophobic derivatization will enhance the hydrophobicity of methylated peptides (the retention time of peptides in reversed-phase chromatography, the shorter the retention time, the stronger the hydrophilicity of the peptide, the longer the retention time, the stronger the hydrophobicity of the peptide), thereby increasing the retention time of methylated peptides in reversed-phase chromatography and improving the identification efficiency of methylated peptides; after enzymatic hydrolysis of the protein solution of the biological sample, the obtained protease hydrolysate is subjected to hydrophobic derivatization modification (preferably hydrophobic modification based on alkyl aldehydes), and the methylated peptides of the control group and the derivatization modification group are enriched respectively (preferably the enrichment method of strong cation exchange chromatography), and the obtained control group and the derivatization modification group are separated. The methylated peptides of the derivatization group were analyzed by mass spectrometry respectively; compared with the control group, the hydrophobicity (retention time of peptides in reverse phase chromatography, the shorter the retention time, the stronger the hydrophilicity of the peptide, the longer the retention time, the stronger the hydrophobicity of the peptide) of the methylated peptides identified in the derivatization modification group was significantly enhanced; hydrophobic derivatization modification can significantly improve the identification of short-chain methylated peptides (peptide length less than 15 amino acid residues); the methylation sites identified by the control group and the derivatization modification group are highly complementary (the methylation sites jointly identified by the control group and the derivatization modification group are approximately twice that of the control group), and the combined analysis of the methylation sites identified by the control group and the derivatization modification group can significantly improve the identification coverage of the methylated proteome.
[0022] The present invention utilizes hydrophobic derivatization modification to enhance the hydrophobicity of methylated peptides and thereby increase the retention time of methylated peptides in reverse phase chromatography. After enzymatic hydrolysis of the protein solution of the biological sample, the obtained protease hydrolysate is subjected to hydrophobic derivatization modification, and the methylated peptides of the control group and the derivatization modification group are respectively enriched, and finally the obtained methylated peptides of the control group and the derivatization modification group are respectively subjected to mass spectrometry analysis. After hydrophobic derivatization, the identification of short-chain methylated peptides (peptide length less than 15 amino acid residues) will be improved; and the methylation sites identified by the control group and the derivatization modification group are highly complementary (the methylation sites jointly identified by the control group and the derivatization modification group are approximately twice that of the control group), and the combined analysis of the methylation sites identified by the control group and the derivatization modification group can effectively improve the coverage of methylated proteome identification.
[0023] Advantages of the present invention:
[0024] Methylated peptides have strong hydrophilicity and are weakly retained in reversed-phase chromatography, resulting in low identification efficiency of methylated peptides. Hydrophobic derivatization will enhance the hydrophobicity of methylated peptides and increase the retention time in reversed-phase chromatography, thereby improving the identification efficiency of methylated peptides. Hydrophobic derivatization will improve the identification of short-chain methylated peptides (peptide length less than 15 amino acid residues). In addition, the methylation sites identified by the control group and the derivatization modification group are highly complementary (the methylation sites identified jointly by the control group and the derivatization modification group are approximately twice that of the control group). Combined analysis of the methylation sites identified by the control group and the derivatization modification group can effectively improve the coverage of methylated proteome identification. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The flowchart of the derivatization strategy for identifying protein methylation modification is shown in FIG. The protein solution of the biological sample is enzymatically hydrolyzed by protease, and then the obtained peptides are derivatized by a hydrophobic derivatization strategy, and the methylated peptides of the control group and the derivatization modification group are enriched respectively, and the methylated peptides of the control group and the derivatization modification group are analyzed by LC-MS / MS respectively;
[0026] Figure 2 Comparison of derivatization efficiency between one reaction and two reactions for hydrophobic modification of alkyl aldehydes;
[0027] Figure 3 The derivatization strategy is used to identify and analyze protein methylation modification, (A) the retention time of the methylated peptides identified in the control group; (B) the retention time of the methylated peptides identified in the derivatization modification group; (C) the distribution of methylated peptides of different chain lengths identified in the control group and the derivatization modification group;
[0028] Figure 4The derivatization strategy is used to identify and analyze protein methylation modifications, (A) the distribution of methylation forms, methylation sites, and methylated proteins identified by the control group and the derivatization modification group; (B) the distribution of lysine and arginine methylation forms identified by the control group and the derivatization modification group; (C) the methylation sites identified by the control group and the derivatization modification group; (D) the monomethylated arginine and dimethylated arginine identified by the control group and the derivatization modification group; (E) the monomethylated lysine, dimethylated lysine, and trimethylated lysine identified by the control group and the derivatization modification group;
[0029] Figure 5 For the identification and analysis of protein methylation modification by the derivatization strategy, the methylation forms collected in the Phosphositeplus database represent known methylation forms, short-chain methylation forms represent amino acid residues less than 15, and long-chain methylation forms represent amino acid residues greater than or equal to 15;
[0030] Figure 6 The derivatization strategy was used to identify and analyze protein methylation modifications: (A) Motif analysis of arginine methylation; (B) Motif analysis of lysine methylation. DETAILED DESCRIPTION
[0031] This example is based on the identification of improved protein methylation by hydrophobic derivatization of alkyl aldehydes. The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.
[0032] Example
[0033] The specific steps of the method for improving protein methylation modification identification and analysis are as follows:
[0034] (1) 1×10 7 NCI-H460 lung cancer cells were cultured in 8 mL of 15 mg / L heavy-labeled methionine (L-methionine-(methyl- 13 C,D 3 ))(Sigma, 299154)RPMI-1640 medium (Thermo, A1451701, lacking methionine), the cells were cultured in this medium for 8 generations (one generation is one cell split into two cells), so that the intracellular L-methionine-(methyl-CH 3 ) is completely converted into L-methionine-(methyl- 13 C,D 3); Wash three times with 1 mL 10 mM PBS buffer (pH 7.4), add 1 mL lysis buffer solution (8 M urea, 50 mM tris(hydroxymethyl)aminomethane, pH 7.4, 65 mM dithiothreitol, add 1% polyethylene glycol octylphenyl ether and 1% protease inhibitor cocktail (Sigma, p8340) in water);
[0035] (2) Ultrasonication was performed at 150 W for 10 cycles, each cycle for 6 s, and centrifuged at 20,000 g for 10 min to obtain a cell lysate containing protein; dithiothreitol with a final concentration of 20 mM was added to two portions of cell lysate containing 200 μg of protein (a total of 2 portions of cell lysate, each containing 200 μg of protein), and the mixture was incubated in a 37°C water bath for 2 h, and then iodoacetamide with a final concentration of 40 mM was added, and the mixture was reacted at 25°C in the dark for 40 min;
[0036] (3) Ultrafiltration and centrifugation were performed at a centrifugal force of 14000 g using an ultrafiltration tube with a molecular weight cutoff of 10K (Sartorius, VN01H02). Each of the two cell lysates was washed three times with 200 μL of 50 mM tris(hydroxymethyl)aminomethane buffer solution at a pH of 8, and 200 μL of 50 mM tris(hydroxymethyl)aminomethane buffer solution at a pH of 8 was added to an ultrafiltration tube (Sartorius, VN01H02). Then, trypsin (Sigma, T1426) and lysine enzyme Lys-C (Beijing Enzyme Source Biotechnology Co., Ltd., P01002) were added at a protein weight ratio of 1:50, and enzymatic hydrolysis was carried out in a 37°C water bath for 16 h.
[0037] (4) Ultrafiltration centrifugation was performed at a centrifugal force of 14000 g using an ultrafiltration tube with a molecular weight cutoff of 10K. The filtrate was the two enzymatic hydrolysates obtained. 20 μL of a 50 mM tris(hydroxymethyl)aminomethane activation buffer solution containing 5 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetic acid, 5 mM calcium chloride and a pH value of 7.6 was added to the two enzymatic hydrolysates obtained. Then, arginase Arg-C (Shanghai Union Biotech Co., Ltd., LS001641) was added to the two enzymatic hydrolysates at a protein weight ratio of 1:50. The hydrolysates were enzymolyzed in a 37°C water bath for 16 h to obtain two enzymatic hydrolysates of three enzymatic hydrolysates.
[0038] (5) Add trifluoroacetic acid to the obtained two portions of the three enzyme-digested protein hydrolysates to adjust the pH value to 2-3 (here 2.6), activate the C18 solid phase extraction column (Waters, 60 mg, WAT094226) with 600 μl of methanol, wash away the methanol with 600 μl of a final volume concentration of 0.1% trifluoroacetic acid aqueous solution, add the three enzyme-digested protein hydrolysates to the C18 solid phase extraction column (Waters, 60 mg, WAT094226), wash with 600 μl of a final volume concentration of 0.1% trifluoroacetic acid aqueous solution, and then elute with 600 μl of a final volume concentration of 80% acetonitrile / 0.1% trifluoroacetic acid aqueous solution, freeze-dry the obtained peptide eluates, and obtain two portions of enzymatic peptides of the control group;
[0039] (6) Take one portion of the enzymatic peptide fragment from the control group and redissolve it in 200 μL of a reducing alkylation buffer solution (100 mM butyraldehyde, 200 mM pyridine and 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 8, in water) to make the final concentration of the peptide fragment 1 μg / μL, and perform the first reaction in a 25°C water bath for 2 h; then add 200 μL of a reducing alkylation buffer solution containing 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 8, 200 mM butyraldehyde and 200 mM pyridine to the peptide fragment solution of the first reaction, and continue the reaction in a 25°C water bath for 16 h to obtain the protease hydrolyzate of the derivatization modification group;
[0040] (7) Add trifluoroacetic acid to the protease hydrolysate of the derivatization modification group obtained in step (6) to adjust the pH to 2-3 (here 2.6), activate a C18 solid phase extraction column (Waters, 60 mg, WAT094226) with 600 μl of methanol, wash away the methanol with 600 μl of a final volume concentration of 0.1% trifluoroacetic acid aqueous solution, add the protease hydrolysate to a C18 solid phase extraction column (Waters, 60 mg, WAT094226), then wash with 600 μl of a final volume concentration of 0.1% trifluoroacetic acid aqueous solution, and then elute with 600 μl of a final volume concentration of 80% acetonitrile / 0.1% trifluoroacetic acid aqueous solution, freeze-dry the obtained peptide eluate, and obtain the enzymatic peptides of the derivatization modification group;
[0041] (8) Put the absorbent cotton (1 mm in diameter and 1 cm in height) into the tip of a 200 μL pipette tip (Thermo, 94300320, Aibende, 2244010002, Ruining, RT0200-RC-FS), and use a capillary quartz tube (Polymicro Technologies, UVQ04B, inner diameter: 150 μm) to compact the absorbent cotton as a sieve plate (about 0.5 cm in length), named tips; wash the tips once with 200 μL acetonitrile, add 100 μL acetonitrile to the tips, and add 60 μL strong cation exchange chromatography material (Suzhou Saifen Technology Co., Ltd., RM0310722, functional group: methyl sulfonate, 30 μm, ) were added to 200 μL of tips, and centrifuged at 500 g for 1 min; the mixture was washed once with 200 μL of 5 mM Brønsted-Rodriguez (5 mM acetic acid, 5 mM phosphoric acid and 5 mM boric acid in water) pH 12 buffer solution I, and then washed twice with 200 μL of 5 mM Brønsted-Rodriguez (5 mM acetic acid, 5 mM phosphoric acid and 5 mM boric acid in water) pH 2 buffer solution II, and equilibrated twice with 200 μL of equilibrated buffer solution (volume ratio, 50% acetonitrile, 50% 5 mM Brønsted-Rodriguez (5 mM acetic acid, 5 mM phosphoric acid and 5 mM boric acid in water), pH 2);
[0042] (9) The enzymatic peptide fragments of the control group obtained in step (5) and the derivatized modified group obtained in step (7) were respectively redissolved in 400 μL of balanced buffer aqueous solution (volume ratio, 50% acetonitrile, 50% 5 mM Bromide (5 mM acetic acid, 5 mM phosphoric acid and 5 mM boric acid, in water), pH 2), and added to the 200 μL tips obtained in step (8);
[0043] The enzymatic peptides of the derivatized group were washed three times with 200 μL of washing buffer solution III (volume ratio, 85% acetonitrile, 15% 5 mM Boron-Rodriguez (5 mM acetic acid, 5 mM phosphoric acid and 5 mM boric acid, in water), pH 9); the enzymatic peptides of the control group were washed three times with 200 μL of washing buffer solution IV (volume ratio, 80% acetonitrile, 20% 5 mM Boron-Rodriguez (5 mM acetic acid, 5 mM phosphoric acid and 5 mM boric acid, in water), pH 9);
[0044] The enzymatic peptides of the derivatization modification group and the enzymatic peptides of the control group were eluted twice with 200 μL of elution buffer solution (equal volumes of acetonitrile and 10% ammonia water), and the peptide eluates obtained were freeze-dried to obtain methylated peptides of the control group and the derivatization modification group respectively;
[0045] (10) The methylated peptides from the control group and the derivatization modification group were respectively redissolved in 20 μL of 0.1% trifluoroacetic acid for RP LC-MS / MS analysis.
[0046] (11) The raw files collected by mass spectrometry were searched using the Maxquant search engine (Version 1.5.3.30) to obtain the peptide information of the control group and the derivatization modification group; compared with the butyraldehyde derivatization modification of the single reaction, the N-terminus and the primary amine group of the peptide in the double reaction were almost all reacted with two aldehyde groups, indicating that the hydrophobic derivatization modification efficiency of the present invention is relatively high ( Figure 2 ); Compared with the control group, the retention time of the methylated peptides identified in the derivatization modification group was significantly increased ( Figure 3 A-3B), and the derivatization modification group identified more short-chain methylated peptides (peptide length less than 15 amino acid residues), such as Figure 3 As shown in C, it shows that the derivatization modification enhances the hydrophobicity of the methylated peptides and thus improves the identification efficiency of the methylated peptides; the control group only identified 356 methylation sites, while the derivatization modification group identified 492 methylation sites ( Figure 4 A), and the methylation sites identified by the control group and the derivatization modification group were highly complementary (188 methylation sites were identified together, and a total of 660 methylation sites were identified), such as Figure 4 As shown in C, combining the methylation sites identified in the control group and the derivatization modification group can significantly improve the identification coverage of the methylated proteome.
[0047] Figure 2 The derivatization efficiency of two reactions of butyraldehyde hydrophobic modification was significantly higher than that of one reaction. The ratio of two aldehyde groups on the N-terminus of the peptide and the primary amine group of lysine in the two reactions was significantly higher than that in the one reaction. Only one reaction was performed before the optimization conditions, and two reactions were performed after the optimization conditions.
[0048] Figure 3 Hydrophobic derivatization modification can significantly improve the identification efficiency of methylated peptides. The retention time of methylated peptides identified by the derivatization modification group is significantly increased ( Figure 3 A-3B), and significantly improves the identification of short-chain methylated peptides (peptide length less than 15 amino acid residues) ( Figure 3 C), Native represents the control group, and Derivatized represents the derivatized modification group.
[0049] Figure 4Hydrophobic derivatization modification can enhance the identification coverage of the methylated proteome. A total of 356 methylation sites were identified in the control group, corresponding to 411 methylation forms (arginine methylation includes monomethylation (MMA) and dimethylation (DMA), lysine methylation includes monomethylation (Kme1), dimethylation (Kme2) and trimethylation (Kme3)), while a total of 492 methylation sites were identified in the derivatization modification group, corresponding to 586 methylation forms (arginine methylation includes monomethylation (MMA) and dimethylation (DMA), lysine methylation includes monomethylation (Kme1), dimethylation (Kme2) and trimethylation (Kme3)). Figure 4 As shown in A; Derivatization modification can improve the identification of arginine methylation sites ( Figure 4 B); the methylation sites identified by the control group and the derivatization modification group were highly complementary (188 methylation sites were identified together, and a total of 660 methylation sites were identified), such as Figure 4 C, and 119 arginine methylation sites have two methylation forms (arginine monomethylation and dimethylation), as shown in Figure 4 As shown in D, there are only two lysine methylation sites with three methylation forms (lysine monomethylation, dimethylation, and trimethylation). Figure 4 As shown in E, Native represents the control group, and Derivatized represents the derivatized modification group.
[0050] Figure 5 Hydrophobic derivatization modification can identify more new methylation forms (arginine methylation includes monomethylation (MMA) and dimethylation (DMA), lysine methylation includes monomethylation (Kme1), dimethylation (Kme2) and trimethylation (Kme3)). The methylation forms of short peptides (arginine methylation includes monomethylation (MMA) and dimethylation (DMA), lysine methylation includes monomethylation (Kme1), dimethylation (Kme2) and trimethylation (Kme3)) increased from 62 to 181, while the methylation forms of long peptides (arginine methylation includes monomethylation (MMA) and dimethylation (DMA), lysine methylation includes monomethylation (Kme1), dimethylation (Kme2) and trimethylation (Kme3)) were slightly reduced. Native represents the control group and Derivatized represents the derivatized modification group.
[0051] Figure 6 The motif analysis results of the identified protein methylation, the motif of the arginine methylation site is RG / RGG ( Figure 6A), which is consistent with the previously reported arginine methylation motif (Reference 4. Uhlmann, T., Geoghegan, VL, Thomas, B., Ridlova, G., Trudgian, DC, Acuto, OA method for large-scale identification of protein arginine methylation. Molecular & Cellular Proteomics, 2012, 11 (11), 1489-1499.), indicating that the identified methylation sites are highly reliable.
[0052] In summary, the present invention is based on the hydrophobic derivatization strategy of alkyl aldehydes for identifying protein methylation modification. Derivatization modification can significantly improve the identification of short-chain methylated peptides (peptide length less than 15 amino acid residues); and significantly improve the identification of arginine methylation; combined analysis of the control group and the derivatization modification group jointly identified methylation sites is about twice that of the control group. Therefore, the present invention is a method for identifying and analyzing protein methylation modification with great application prospects.
Claims
1. A method for improving protein methylation modification identification and analysis, characterized in that: The protease hydrolysate of the biological sample is subjected to hydrophobic derivatization modification, and the methylated peptides in the protease hydrolysate of the control group and the protease hydrolysate of the derivatization modification group are enriched respectively. The methylated peptides of the control group and the derivatization modification group are subjected to mass spectrometry analysis respectively. By combining the analysis of the methylation sites identified in the control group and the derivatization modification group, the site information of protein methylation in the biological sample can be obtained.
2. The method according to claim 1, characterized in that The specific steps of this method are: (1) enzymatically hydrolyzing a protein solution of a biological sample into peptide fragments to obtain a protease hydrolysate; The peptides in the obtained protease hydrolysate are subjected to hydrophobic derivatization modification by using a hydrophobic derivatization strategy, and the protease hydrolysate in which the peptides are subjected to hydrophobic derivatization modification serves as a derivatization modification group; The protease digest of the peptides without derivatization modification was used as the control group; (2) Enriching the methylated peptides in the protease hydrolysates of the control group and the derivatization modification group respectively; (3) Using mass spectrometry to analyze the methylated peptides in the control group and the derivatization modification group; (4) Using search software to search the files generated by mass spectrometry, the methylation sites of the control group and the derivatization modification group were obtained respectively; By combining the methylation sites identified in the control group and the derivatization modification group, the site information of protein methylation in the biological sample can be obtained.
3. The method according to claim 2, characterized in that The protein solution of the biological sample includes one or more of cell and / or tissue extracts; the cell and / or tissue extracts are derived from one or more of cells and / or tissues in animals, plants, cells or humans; The biological sample protein solution is obtained by lysing tissues and / or cells using a buffer, so that the protein solution in the tissues and / or cells is denatured; A preferred buffer contains 1-10M urea, 10-100mM dithiothreitol and 5-100mM tris(hydroxymethyl)aminomethane solution with a volume concentration of 0.5-2% polyethylene glycol octylphenyl ether, and the tissues and / or cells are ultrasonically disrupted and lysed in the buffer; A preferred protein solution of tissues and / or cells is prepared by metabolically labeling L-methionine-(methyl-CH3) in tissues and / or cells into L-methionine-(methyl- 13 C, D3), which helps to improve the credibility of protein methylation identification.
4. The method according to claim 2 or 3, characterized in that: The proteolysis process is carried out in two steps. Step 1: The proteases used are any two proteases with different cleavage sites among trypsin, lysinase Lys-C, arginase Arg-C, Glu-C protease, pepsin, etc., but are not limited to these proteases; the usage ratio of the two proteases with different cleavage sites to the protein is 1:10-1:100 (mass ratio of protease to protein to be hydrolyzed); Using two proteases with different cleavage sites (the ratio of the amount of protease to the protein to be hydrolyzed is 1:10-1:100, preferably 1:50) to perform a first enzymatic hydrolysis on the protein solution of tissues and / or cells, such as Glu-C protease (specifically hydrolyzes the carboxyl terminal peptide bonds of glutamic acid and aspartic acid) and pepsin (specifically hydrolyzes aromatic amino acids, such as one or more of the carboxyl terminal peptide bonds of phenylalanine, tyrosine, leucine and tryptophan), to obtain two enzymatic hydrolyzates; Step 2: using a protease with a different cleavage site from the first two proteases (the ratio of the protease to the protein to be hydrolyzed is 1:10-1:100, preferably 1:50) to further hydrolyze the protease hydrolyzate obtained by two enzymes, such as lysinase Lys-C (specifically hydrolyzes the peptide bond at the carboxyl terminal of lysine), to obtain a protease hydrolyzate obtained by three enzymes; Through two-step protease hydrolysis, shorter enzymatic peptides are obtained, which is conducive to hydrophobic derivatization modification (the peptides are long, and the hydrophobicity of the peptides will become stronger after hydrophobic derivatization modification, which will have a strong retention in reversed-phase chromatography and be difficult to be eluted, resulting in a decrease in the identification efficiency of methylated peptides); Through two-step protease hydrolysis, the missed cleavage rate of peptides is effectively reduced, which is beneficial to the subsequent separation and enrichment of methylated peptides.
5. The method according to claim 2, characterized in that: The hydrophobic derivatized group may be one or more of the thiol group of cysteine, the carboxyl group of the peptide segment, the amino group of the peptide segment, etc., but is not limited to these groups; The thiol group of modified cysteine can be one or more of N,N-dimethyl-2-chloroethylamine, iodoacetanilide, N-ethylmaleimide, etc., but is not limited to these reaction reagents; The carboxyl group can be modified by one or more of 1-(2-pyrimidine)piperazine, 2-p-nitrophenylhydrazine, benzylamine, 1-benzylpiperazine, etc., but is not limited to these reaction reagents; The amino group can be modified with one or more of coumarin derivatives, sulfonyl chloride, alkyl aldehyde, diisopropyl phosphite, etc., but is not limited to these reaction reagents.
6. The method according to claim 5, characterized in that: The preferred derivatizing agent is an alkyl aldehyde that modifies the amino group, and the alkyl aldehyde includes one or more of acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, hexanal, etc., but is not limited to these aldehydes; The derivatization process is as follows: using a buffer solution containing 50-200 mM alkyl aldehyde and 50-200 mM pyridine and an enzymatic peptide fragment with a final concentration of 0.1-10 μg / μl to react for the first time in a water bath at 20-30°C for 1-6 hours; then adding a buffer solution containing 100-500 mM alkyl aldehyde and 100-500 mM pyridine to the solution of the first reaction, and performing a second reaction in a water bath at 20-30°C for 1-24 hours; the volume ratio of the buffer solution added for the second time to the solution of the first reaction is 0.5:1-3:1; Due to the steric hindrance of the aldehyde group, the N-terminus of most peptides and the primary amine group of lysine react with only one aldehyde group in a derivatization reaction. In order to make the N-terminus of each peptide and the primary amine group of lysine react with two aldehyde groups, a second derivatization reaction was performed to enhance the hydrophobicity of the peptides, which was beneficial to improve the identification of methylated peptides. A preferred buffer is 10-100 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer.
7. The method according to claim 2, 5 or 6, characterized in that: The obtained peptides were subjected to hydrophobic derivatization modification using a hydrophobic derivatization strategy as a derivatization modification group; the peptides that were not subjected to derivatization modification were used as a control group; The methylated peptides of the control group and the derivatization modification group are enriched by chromatography, antibodies, recognition domains, etc., but are not limited to the above methods; The chromatographic method includes one or more of affinity chromatography, strong cation exchange chromatography, strong anion exchange chromatography, etc., but is not limited to these chromatographic methods; The antibody method includes one or more of lysine methylation antibodies, arginine methylation antibodies, etc., but is not limited to these antibodies; The recognition domains include one or more of MBT domains (malignant brain tumor domains), Tudor domains, PHD domains (plant homeodomain fingers), chromatin domains (chromodomains), PWWP domains (proline-tryptophan-tryptophan-proline domains), WD40 domains (WD40 repeat domains), BAH domains (bromo adjacent homology domains) and ANK domains (ankyrin repeats), but are not limited to these domains; The preferred method for separating and enriching the methylated peptides in the control group and the derivatized modified group is strong cation exchange chromatography, including one or more cation exchange groups such as methyl sulfonate and carboxymethyl, but not limited to these exchange groups; The separation and enrichment process is as follows: 1-200 μL of strong cation exchange chromatography material is added to tips (solid phase extraction pipette tips / gun tips, using the absorbent cotton loaded at the tip of the gun tip as a sieve plate), washed 1-3 times with 100-1000 μL of washing buffer I, then washed 1-3 times with 100-1000 μL of washing buffer II, equilibrated 1-3 times with 100-1000 μL of equilibration buffer, and the obtained enzymatic peptides of the control group and the derivatization modification group are respectively redissolved in 100-1000 μL of equilibration buffer. , respectively added to the tips containing strong cation exchange chromatography materials (solid phase extraction pipette tip / gun tip, using the absorbent cotton loaded in the tip of the gun tip as a sieve plate), using 100-1000 μL of washing buffer III to wash the enzymatic peptides of the derivatization modification group 1-3 times, using 100-1000 μL of washing buffer IV to wash the enzymatic peptides of the control group 1-3 times, and using 100-1000 μL of elution buffer to elute 1-3 times, respectively, freeze-drying the obtained peptide eluates, and obtaining methylated peptides of the control group and the derivatization modification group, respectively; Wash buffer I was 1-10 mM Brombe-Rodriguez (1-10 mM acetic acid, 1-10 mM phosphoric acid and 1-10 mM boric acid), pH 10-14; Wash buffer II is 1-10 mM Brombe-Rodriguez (1-10 mM acetic acid, 1-10 mM phosphoric acid and 1-10 mM boric acid), pH 1-5; The equilibration buffer is 10-60% acetonitrile by volume, 40-90% 1-10 mM Bromide (1-10 mM acetic acid, 1-10 mM phosphoric acid and 1-10 mM boric acid) by volume, and the pH value is 1-5; Washing buffer III is 80-100% acetonitrile by volume, 0-20% 1-10 mM Bromide (1-10 mM acetic acid, 1-10 mM phosphoric acid and 1-10 mM boric acid) by volume, and pH is 1-10; Wash buffer IV is 70-100% acetonitrile by volume, 0-30% 1-10 mM Bromide (1-10 mM acetic acid, 1-10 mM phosphoric acid and 1-10 mM boric acid) by volume, and pH is 1-10; The elution buffer is a mixed solution of acetonitrile with a volume content of 10-60% and ammonia water with a mass concentration of 1-50% with a volume content of 40-90%.
8. The method according to claim 2, characterized in that: The methylated peptides obtained in the control group and the derivatization modification group were respectively redissolved in trifluoroacetic acid with a volume concentration of 0.05-1% for RP LC-MS / MS analysis.
9. The method according to claim 2, characterized in that: The obtained mass spectrum files are searched respectively using one or more software such as MaxQuant, pFind, etc., but the search software is not limited to these.
10. The method according to claim 2, characterized in that The method is based on the fact that the weak retention of methylated peptides in reversed-phase chromatography leads to low identification efficiency of methylated peptides, and hydrophobic derivatization will enhance the hydrophobicity of methylated peptides (retention time of peptides in reversed-phase chromatography, the shorter the retention time, the stronger the hydrophilicity of the peptide, the longer the retention time, the stronger the hydrophobicity of the peptide), thereby increasing the retention time of methylated peptides in reversed-phase chromatography and improving the identification efficiency of methylated peptides; after enzymatic hydrolysis of the protein solution of the biological sample, the obtained protease hydrolysate is subjected to hydrophobic derivatization modification (preferably hydrophobic modification based on alkyl aldehydes), and the methylated peptides of the control group and the derivatization modification group are enriched respectively (preferably the enrichment method of strong cation exchange chromatography), and the obtained methylated peptides of the control group and the derivatization modification group are subjected to mass spectrometry analysis respectively; Compared with the control group, the hydrophobicity of the methylated peptides identified in the derivatization modification group (the retention time of the peptide in reversed-phase chromatography, the shorter the retention time, the stronger the hydrophilicity of the peptide, the longer the retention time, the stronger the hydrophobicity of the peptide) was significantly enhanced; Hydrophobic derivatization can significantly improve the identification of short-chain methylated peptides (peptide length less than 15 amino acid residues); The methylation sites identified by the control group and the derivatization modification group were highly complementary (the methylation sites identified by the control group and the derivatization modification group were approximately twice that of the control group). Combining the analysis of the methylation sites identified by the control group and the derivatization modification group can significantly improve the identification coverage of the methylated proteome.