A method for enriching arginine dimethylated peptides based on boron affinity chromatography

Abstract

This invention relates to a method for enriching arginine dimethylated peptides based on boric acid affinity chromatography. First, diketone compounds are used to selectively block unmodified and monomethylated guanidinium groups on the arginine side chains in a whole protein enzymatic digest. Then, an ortho-dicarbonyl compound and boron affinity chromatography material are simultaneously added to the peptide solution. The boron affinity chromatography material selectively captures the cis-ortho-diol intermediate generated by the reaction of the ortho-dicarbonyl compound with dimethylated arginine residues, thereby enriching the arginine dimethylated peptides. This method, combined with mass spectrometry detection and software analysis, allows for large-scale identification of arginine dimethylation modification sites in proteins. Compared with traditional immunoaffinity enrichment methods, this invention has advantages such as high enrichment specificity, good identification effect, and low experimental cost.

Description

Technical Field

[0001] This invention belongs to the field of methylated proteomics technology in proteomics research, specifically involving a method for enriching arginine dimethylated peptides. Technical Background

[0002] Arginine methylation is a crucial post-translational modification of proteins, playing a vital role in the regulation of many physiological processes, such as RNA processing, transcriptional regulation, signal transduction, DNA repair, and protein-protein interactions. Although arginine methylation has significant regulatory effects on organisms, it has limited impact on the fundamental physicochemical properties of the arginine side chain, making efficient enrichment of arginine-methylated peptides challenging. There are three main types of arginine methylation: monomethylation, asymmetric dimethylation, and symmetric dimethylation. A series of antibodies have been developed to target these three types of methylation modifications (Geoghegan V. et al. Nature communications, 2015, 6(1):1-8; Musiani D. et al. Science signaling, 2019, 12(575):eaat8388.). However, antibodies are more effective at enriching arginine monomethylated peptides than arginine dimethylated peptides. Furthermore, antibody-based enrichment methods require a protein sample volume of ten milligrams per experiment; simultaneously, antibodies are very expensive, and batch-to-batch reproducibility in antibody production is often problematic. Therefore, it is crucial to develop a low-cost and effective method for enriching arginine dimethylated peptides.

[0003] In 2003, Lindner et al. (Leitner A. et al. Journal of mass spectrometry, 2003, 38(8): 891-899.) reported that arginine residues can react with 2,3-butanedione to form a cis-ortho-dihydroxy structure, which can then react with boric acid to form a stable five-membered ring structure. Utilizing this principle, their research group (Foettinger A. et al. Journal of Chromatography A, 2005, 1079(1-2): 187-196.) successfully enriched arginine-containing peptides using boron affinity chromatography. Besides butanedione, arginine residues can also specifically react with 1,2-cyclohexanedione (Smith ELMethods in enzymology. Academic Press, 1977, 47: 156-161.) to generate an irreversible product.

[0004] Based on the different reactivity of arginine residues with different degrees of methylation, this invention uses diketone compounds (such as 1,2-cyclohexanedione) to block unmodified and monomethylated arginine residues. Then, it utilizes the reaction between vicinal dicarbonyl compounds and the guanidinium group of the dimethylated arginine side chain to form a cis-vicinal diol structure, and boron affinity chromatography to enrich compounds containing this structure. This invention is the first to develop a highly selective method for enriching dimethylated arginine peptides, effectively overcoming the problems existing in the antibody enrichment process. Summary of the Invention

[0005] The purpose of this invention is to provide a method for enriching arginine dimethyl peptides based on dicarbonyl compounds, boron affinity chromatography, and ortho-dicarbonyl compounds, which has good enrichment specificity and high enrichment recovery rate, and overcomes the disadvantages of antibody-based immunoaffinity enrichment strategies, such as excessive sample usage and high experimental costs.

[0006] This invention discloses a method for enriching arginine dimethylated peptides based on boron affinity chromatography. The method uses diketone compounds to selectively react with guanidinium groups on the arginine side chains of protein-digested peptides or peptide mixtures, both unmodified and modified with a single methyl group, thereby reducing the chemical reactivity of these two types of guanidinium groups. Then, ovoid dicarbonyl compounds are used to specifically modify a cis-ovoid diol group onto the guanidinium group on the arginine side chain modified with two methyl groups. Simultaneously, chromatographic materials modified with boric acid groups are used to capture this cis-ovoid diol group, thus achieving the specific enrichment of arginine dimethylated peptides.

[0007] 1. The process of obtaining protein hydrolysate peptides in the method includes enzymatic hydrolysis of protein samples to obtain a protein hydrolysate; adding an amino blocking reagent for reaction; desalting and drying to obtain a peptide mixture;

[0008] The specific process is as follows:

[0009] (a) 1 mg of protein sample was dispersed in 0.2 to 2 mL of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer solution or triethylamine carbonate buffer solution with a molar concentration of 10 to 200 mM and a pH of 7.0 to 9.0; the protein sample dispersed in the buffer solution was enzymatically digested with protease to obtain protease hydrolysate.

[0010] (b) An amino blocking reagent was added to the protease hydrolysate for reaction; after the reaction, the sample was desalted and then freeze-dried using a vacuum freeze-drying method to obtain peptide mixture A.

[0011] 2. The protein sample source in step 1-(a) of the method includes one or more of animal cells, plant cells, animal tissues, plant tissues or other sources; preferably, the protein sample is derived from laboratory-cultured animal cancer cells;

[0012] The protease mentioned in step 1-(a) is one or more proteases selected from trypsin, lysine endopeptidase, clostridium protease, Staphylococcus aureus V8 protease, chymotrypsin, carboxypeptidase B, etc., but is not limited to one or more of these enzymes. The enzymatic digestion conditions mentioned in step 1-(a) are: the mass ratio of protease to protein sample is between 1:10 and 1:100, the digestion time is 1 to 24 hours, the digestion temperature of carboxypeptidase B is 37 to 55°C, and the digestion temperature of other proteases is 20 to 37°C.

[0013] Preferably, the protein sample is first digested with trypsin, then with clostridium protease, and finally with carboxypeptidase B. The mass ratio of the three proteases to the protein sample is in the range of 1:20-1:100. The reaction temperature for the first two digestion steps is 35-37℃, and the reaction time is 14-16h. The reaction temperature for the third digestion step is 47-50℃, and the reaction time is 1.5-2h.

[0014] 3. The amino blocking reagent in step 1-(b) of the method is a combination of sodium cyanoborohydride and formaldehyde, but is not limited to such reagents that react with amino groups. The reaction conditions in step 1-(b) are as follows: 20-200 μL of sodium cyanoborohydride with a molar concentration of 0.3-1.0 M and 20-200 μL of formaldehyde aqueous solution with a mass concentration of 1-10% are added to the protein hydrolysate obtained in step 1-(a), the reaction temperature is 20-40℃, and the reaction time is 0.2-2 h.

[0015] 4. The desalination step 1-(b) of the method uses a solid-phase microextraction column filled with C18 microspheres or a solid-phase microextraction column filled with HLB packing material developed by Waters.

[0016] 5. Based on methods 1-4, the enrichment steps for arginine dimethylated peptides are as follows:

[0017] (1) Take 100 μg of peptide mixture (A) and redissolve it in 50-100 μL of sodium hydroxide aqueous solution with a molar concentration of 100-400 mM. Add diketone compounds to carry out the reaction. After the reaction is completed, use hydrochloric acid aqueous solution with a concentration of 0.05-12 M to adjust the pH of the solution to about 5-9 to obtain peptide mixture B.

[0018] (2) Add 100-250 μL of 25-50 mM triethylamine carbonate buffer to peptide mixture B, with a pH between 10 and 11. Then add 0.1-1 M sodium chloride aqueous solution until the final sodium chloride concentration is 40-100 mM. Finally, add an ortho-dicarbonyl compound and 5-80 μL of chromatographic material modified with boric acid groups. Incubate at 10-25°C for 0.5-2 h. After the reaction, filter and collect the chromatographic material. Wash the chromatographic material 3-6 times with 5-25 mM triethylamine carbonate buffer, each time using 20-100 μL of solution with a pH between 9.6 and 10.2. Finally, elute the peptides adsorbed on the chromatographic packing material with acidic aqueous solution. Then freeze-dry the eluent using vacuum freeze-drying to obtain crude arginine dimethyl peptides.

[0019] (3) Take 5-20 μL of a solid-phase microextraction column prepared with strong cation exchange chromatography packing material, wash the column with elution buffer 2-6 times, each time using a solution volume of 5-40 μL; then wash the column with loading buffer 2-6 times, each time using a solution volume of 5-40 μL; redissolve the crude extracted arginine dimethyl peptide in 40-100 μL of loading buffer, and load it onto the solid-phase microextraction column, then wash the column with impurity removal buffer 2-6 times, each time using a solution volume of 5-20 μL; finally, elute the peptide adsorbed on the column with 40-100 μL of elution buffer to obtain the elution product; place the elution product incubate at 20-37℃ for 0.4-2 h, and then freeze-dry it to obtain the arginine dimethyl peptide.

[0020] 6. The diketone compound in step 5-(1) of the method is 1,2-cyclohexanedione, but is not limited to this reagent that reacts with the guanidinium group of the arginine side chain without modification or with monomethylation. The reaction conditions in step 5-(1) are as follows: 1,2-cyclohexanedione is added to the reconstituted peptide mixture A until the final concentration of 1,2-cyclohexanedione is 10-40 mM, the reaction temperature is 30-40 °C, and the reaction time is 0.5-2 h.

[0021] 7. The ortho-dicarbonyl compound in step 5-(2) of the method is acetone aldehyde, but is not limited to this reagent that can react with the guanidinium group of the arginine side chain to generate a cis-ortho-diol structure. The specific conditions for the reaction in step 5-(2) are: 1-5 μL of acetone aldehyde aqueous solution with a mass concentration of 10-40% is added to the reaction system.

[0022] The chromatographic packing material modified with boric acid groups in step 5-(2) is a chromatographic material composed of boric acid groups, different spacer arms and agarose material. The type of spacer arms includes one or more of benzene, aminobenzene and carboxybenzene, carbon chains or combinations of groups with a length of 3 to 12 atoms.

[0023] Preferably, the agarose chromatography packing material is modified with m-aminophenylboronic acid.

[0024] 8. The acidic aqueous solution in step 5-(2) of the method includes one or more of the following: a formic acid aqueous solution with a volume concentration of 0.01-0.2%, a trifluoroacetic acid aqueous solution with a volume concentration of 0.01-0.2%, a hydrochloric acid aqueous solution of 1-10mM, a sulfuric acid aqueous solution of 1-10mM, or a nitric acid aqueous solution of 1-10mM, and the volume of the acidic aqueous solution used to elute the peptide fragments is 250-500 μL.

[0025] 9. In the method described above:

[0026] The loading buffer in step 5-(3) is a solution made by mixing an organic solvent and a buffer salt aqueous solution in a certain proportion; the organic solvent type includes one or more reagents such as methanol, ethanol, and acetonitrile, but is not limited to one or more of these organic solvents, and the volume concentration of the organic solvent in the loading buffer is between 10% and 30%; the buffer salt type in the buffer salt aqueous solution includes one or a combination of potassium dihydrogen phosphate and sodium dihydrogen phosphate, but is not limited to one or more of these buffer salts; the molar concentration of the buffer salt in the aqueous solution is 5 to 15 mM, and the pH of the buffer salt aqueous solution is between 2.0 and 3.0;

[0027] The impurity removal buffer solution described in step 5-(3) is a solution made by mixing an organic solvent and an acidic aqueous solution in a certain proportion; the organic solvent type includes one or more reagent combinations such as methanol, ethanol, and acetonitrile, but is not limited to one or more of these organic solvents, and the volume concentration of the organic solvent in the impurity removal buffer solution is between 10% and 30%; the acid type in the acidic aqueous solution includes trifluoroacetic acid, acetic acid, or formic acid, but is not limited to one or more of these acids, and the volume concentration of the acid in the acidic aqueous solution is between 0.005% and 0.1%;

[0028] The elution buffer in step 5-(3) is a solution made by mixing an organic solvent and an alkaline aqueous solution in a certain proportion; the organic solvent type includes one or more reagent combinations such as methanol, ethanol, and acetonitrile, but is not limited to one or more of these organic solvents, and the volume concentration of the organic solvent in the elution buffer is between 10% and 30%; the type of base in the alkaline aqueous solution includes ammonia, monomethylamine, dimethylamine, trimethylamine or triethylamine, but is not limited to one or more of these bases, and the mass concentration of the base in the alkaline aqueous solution is between 0.5% and 2%.

[0029] This method, combining mass spectrometry detection and software analysis, enables large-scale identification of arginine dimethylation modification sites in proteins. Compared with traditional immunoaffinity enrichment methods, this invention offers advantages such as high enrichment recovery, high enrichment specificity, good identification results, and low experimental cost.

[0030] This invention has the following characteristics:

[0031] 1. The amount of protein sample required for the experiment is very low. Compared to immunoaffinity enrichment, which requires up to 10 mg of protein sample, this method requires less than 0.5 mg of protein.

[0032] 2. High enrichment specificity. When analyzing the enriched peptides using liquid chromatography-mass spectrometry (LC-MS), the number of arginine dimethylated peptides identified by this method accounts for 20-50% of the total number of peptides identified, while the enrichment specificity of immunoaffinity enrichment is less than 15%.

[0033] 3. It can simultaneously enrich both asymmetric and symmetric arginine dimethylated peptides.

[0034] 4. Low experimental cost. The antibodies used in immunoaffinity enrichment methods are expensive, while the chemical reagents used in this method are inexpensive. Attached Figure Description

[0035] Figure 1 This is an experimental flowchart of the boron affinity chromatography-based enrichment method for arginine dimethylated peptides. CHD is an abbreviation for 1,2-cyclohexanedione.

[0036] Figure 2To test the effectiveness of this method in enriching arginine dimethylated peptides from a mixture of synthetic peptides, four images are MALDI mass spectra of the peptides. Four synthetic peptides with the sequence GGNFSGRGGFGGS were used in this experiment, but the modifications at the seventh arginine residue (R) differed: no modification, monomethylation, asymmetric dimethylation, and symmetric dimethylation, respectively. The N-terminal amino groups of the four peptides were first dimethylated (resulting in mass-to-charge ratios of 1184, 1198, 1212, and 1218 in the MALDI spectra), and then these peptides were mixed in specific ratios. (A) A mixture of unmodified, monomethylated, and asymmetric dimethylated synthetic peptides in a 2:2:1 ratio. (B) A mixture of unmodified, monomethylated, and symmetric dimethylated synthetic peptides in a 2:2:1 ratio. (C) The product obtained after treating the mixture of synthetic peptides described in (A) using this method. (D) The product obtained by treating the synthetic peptide mixture described in (B) using this method.

[0037] Figure 3 To test the effectiveness of this method in enriching arginine dimethylated peptides from trypsin digest of bovine serum albumin. All four figures are MALDI mass spectra of the peptides. After trypsin digestion of bovine serum albumin, it was mixed with synthetic arginine dimethylated peptides at a mass ratio of 100:1, and then all primary amine groups on the peptides were dimethylated. Two synthetic arginine dimethylated peptides were used in this experiment, both with the sequence GGNFSGRGGFGGSR, but the dimethylation modification at the seventh arginine residue differed between the two peptides; one was asymmetric dimethylation, and the other was symmetric dimethylation. After the primary amine groups were dimethylated, the mass-to-charge ratios of the two peptides in the MALDI spectra were 1368 and 1374, respectively. (A) Product obtained by mixing the peptides from bovine serum albumin digestion with the asymmetric arginine dimethylated peptides; all primary amine groups on the peptide mixture were dimethylated. (B) The product obtained by mixing peptides obtained from enzymatic hydrolysis of bovine serum albumin with symmetrical arginine dimethylated peptides, wherein all primary amine groups on the peptide mixture are labeled with dimethyl groups. (C) The arginine dimethylated peptides in the peptide mixture described in (A) are enriched using this method. Since the C-terminus of the arginine dimethylated peptides has no modified arginine residues that react with 1,2-cyclohexanedione, the final mass-to-charge ratio of the asymmetric arginine dimethylated peptides in the MALDI spectrum is 1462. (D) The arginine dimethylated peptides in the peptide mixture described in (B) are enriched using this method. Since the C-terminus of the arginine dimethylated peptides has no modified arginine residues that react with 1,2-cyclohexanedione, the final mass-to-charge ratio of the symmetrical arginine dimethylated peptides in the MALDI spectrum is 1468.

[0038] Figure 4 The figure shows the number of arginine dimethylation sites in Jurkat T cells identified using different methods. In addition to this method, the figure also compares two antibody-based enrichment methods, which can recognize asymmetric dimethylarginine (aDMA) and symmetric dimethylarginine (sDMA), respectively. The RG and RGG sequence types shown in the figure indicate that the methylation sites are located on the RG and RGG sequences, respectively. Detailed Implementation

[0039] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0040] Example 1

[0041] A boron affinity chromatography-based method for enriching arginine dimethylated peptides in a mixture of synthesized peptides is proposed.

[0042] (1) Take 100 μg of each of the four synthesized peptides (all with the sequence GGNFSGRGGFGGSR, but with different modifications to the seventh arginine residue: no modification, monomethylation, asymmetric dimethylation, and symmetric dimethylation, respectively), and dissolve them in 200 μL of 100 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer (pH 7.65). Then, add 2 μg of carboxypeptidase B to each of the four peptide solutions and incubate at 50 °C for 2 h to remove the C-terminal arginine residue. After enzyme digestion, for the symmetric arginine dimethylated peptide solution, add 8 μL of 4% deuterated formaldehyde aqueous solution and 8 μL of 0.6 M deuterated cyanoborohydride aqueous solution; for the other three peptides, add 8 μL of 4% formaldehyde aqueous solution and 8 μL of 0.6 M cyanoborohydride aqueous solution, and then incubate the four solutions at 25 °C for 1 h. Subsequently, 20 μL of 1M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7.0) was added to each of the four groups of solutions to quench the reaction, and the samples were then desalted and lyophilized.

[0043] (2) The four dimethylated peptides were reconstituted in water to a final concentration of 0.5 μg / μL. 2 μg of the unmodified arginine peptide, 2 μg of the monomethylated arginine peptide, and 1 μg of the asymmetric dimethylated arginine peptide were added to 50 μL of 200 mM sodium hydroxide aqueous solution, followed by the addition of 0.5 M cyclohexanedione to a final concentration of 20 mM. A separate identical reaction solution was prepared, but with the asymmetric dimethylated arginine peptide replaced by the symmetric dimethylated arginine peptide. After mixing, the solution was incubated at 37°C for 40 min. After the reaction was complete, 400 mM hydrochloric acid was added to adjust the pH of the solution to approximately 7.0. Then, 50 μL of 100 mM sodium chloride aqueous solution, 125 μL of 25 mM triethylamine carbonate buffer (pH 10.2-10.4), 1.54 μL of 40% acetone aldehyde solution, and 40 μL of agarose microspheres modified with m-aminophenylboronic acid (Sigma-Aldrich, catalog number A8312) were added. Before addition, the agarose microspheres were washed three times with 0.1% formic acid aqueous solution, each time using a solution volume three times the volume of the microspheres; then washed three times with 10 mM triethylamine carbonate buffer (pH 10.0), each time using a solution volume three times the volume of the microspheres. After mixing the above substances thoroughly, the mixture was incubated at 15°C for 1 hour. After the reaction was complete, the solution was filtered off, and the microspheres were washed with 50 μL of 10 mM triethylamine carbonate buffer (pH 10.0), repeated four times. The peptides adsorbed on the microspheres were eluted with 50 μL of 0.1% formic acid aqueous solution each time, for a total of five elutions. The eluates were then combined and lyophilized. Finally, the microspheres were reconstituted with an alkaline solution (volume ratio: 70% ammonia / 30% acetonitrile, ammonia concentration of 1%) and allowed to stand at room temperature for 1 h.

[0044] (3) The types of peptides in the samples before and after enrichment were detected using MALDI mass spectrometry. The types of samples detected are shown in Figure 2. The detection results are as follows: Figure 2 As shown. Combined with Figure 2 (A) and Figure 2 As shown in (C), this method successfully enriched asymmetric arginine dimethylated peptides from a mixture of peptides containing unmodified and monomethylated arginine residues; and the binding... Figure 2 (B) and Figure 2 As shown in (D), this method successfully enriched symmetrical arginine dimethylated peptides from a mixture of peptides containing unmodified and monomethylated arginine residues.

[0045] Example 2

[0046] A boron affinity chromatography-based method for enriching arginine dimethylated peptides from trypsin hydrolysate of bovine serum albumin.

[0047] (1) Dissolve 100 μg of bovine serum albumin in 100 μL of lysis buffer (containing 100 mM tris(hydroxymethyl)aminomethane-hydrochloric acid, 6 M guanidine hydrochloride, 10 mM tris(2-carboxyethyl)phosphine, and 40 mM chloroacetamide in water, pH 7.4), and heat to 100 °C for 5 min to alkylate all cysteine ​​residues on the protein. After the sample cools, filter the solution using a 10-kDa ultrafiltration tube. After ultrafiltration, retain the protein on the ultrafiltration membrane, discard the filtered solution, and add an equal volume of 100 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid solution (pH 7.9) to the filter membrane. Then, add 1 μg of trypsin to the solution and incubate at 37 °C for 16 h.

[0048] (2) After enzyme digestion, the solution was ultrafiltered again using a 10-kDa ultrafiltration tube, and the filtrate was collected. Two portions of bovine serum albumin trypsin hydrolysate were prepared according to the above method, and 1 μg of each of the two synthetic arginine dimethyl peptides described in Figure 3 was added (both synthetic peptides were untreated). Following the method described in step (1) of Example 1, the primary amine groups of all peptides in the hydrolysate were dimethyl-labeled using formaldehyde and sodium cyanoborohydride (the process and conditions were the same as step (1) of Example 1), and then the hydrolysate was desalted and lyophilized. The labeling reagents used in the hydrolysate containing symmetrical and asymmetrical arginine dimethyl peptides were different; the former used all deuterated reagents, and the latter used all non-deuterated reagents.

[0049] (3) The peptide was reconstituted in 50 μL of 200 mM NaOH aqueous solution, and then the arginine dimethyl peptide was enriched according to the method described in step (2) of Example 1 (the process and conditions are the same as step (2) of Example 1).

[0050] (4) The types of peptides in the samples before and after enrichment were detected using MALDI mass spectrometry. The types of samples detected are shown in Figure 3. The detection results are as follows: Figure 3 As shown. Combined with Figure 3 (A) and Figure 3 As shown in (C), this method can enrich asymmetric arginine dimethylated peptides from the trypsin hydrolysate of bovine serum albumin; and bind to Figure 3 (B) and Figure 3 As shown in (D), this method can enrich symmetrical arginine dimethylated peptides from the trypsin hydrolysate of bovine serum albumin.

[0051] Example 3

[0052] A boron affinity chromatography-based enrichment method for arginine dimethylated peptides was developed for the identification of arginine dimethyl modification sites in Jurkat T cells.

[0053] (1) 250 μL of lysis buffer (6 M guanidine hydrochloride, 100 mM tris(hydroxymethyl)aminomethane-hydrochloric acid, pH = 8.5), 5 μL of 0.5 M tris(2-carboxyethyl)phosphine, and 20 μL of 0.5 M chloroacetamide were added to 1e7 Jurkat T cells, and the mixture was reacted at 100 °C for 5 min. After cooling in an ice bath, the cells were lysed using an ultrasonic cell disruptor. The resulting protease digest was reacted at 100 °C for another 5 min, and then centrifuged at 8000 rpm for 30 min at 4 °C to remove cell debris. An equal volume of 4 °C pure water was added to the supernatant obtained by centrifugation, followed by 8 times the volume of -20 °C acetone. After mixing, the mixture was incubated at -20 °C overnight.

[0054] (2) Centrifuge the protein sample at 2000 rpm for 15 min at 4℃, remove the supernatant, wash the precipitate twice with 80% acetone (at -20℃), and air-dry at room temperature for 10 min to remove the acetone. Add 400 μL of 100 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer (pH = 7.9) to the protein precipitate, then sonicate to disperse it evenly, and add a certain amount of trypsin (protein:enzyme = 50:1) to enzymatically digest the protein at 37℃ for 16 h. After the reaction, the enzymatic hydrolysate was incubated at 100℃ for 10 min to inactivate trypsin. After cooling, 400 μL of buffer (100 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 10 mM dithiothreitol, 0.4 mM ethylenediaminetetraacetic acid, pH 7.65) and 8.5 μL of 0.5 M calcium chloride aqueous solution were added, mixed well, and then clostridium protease (the same amount of enzyme as trypsin) was added. Enzymatic hydrolysis was continued at 37℃ for 16 h. After the enzymatic hydrolysis was completed, 32 μL of 0.5 M chloroacetamide aqueous solution was added, and the reaction was carried out at 100℃ for 10 min to denature the clostridium protease and consume the remaining dithiothreitol. After the solution cooled, carboxypeptidase B (the same amount as trypsin) was added, and enzymatic hydrolysis was carried out at 50℃ for 2 h.

[0055] (3) Add 90 μL of 1M 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid aqueous solution, 80 μL of 0.6M sodium cyanoborohydride aqueous solution, and 80 μL of 4% formaldehyde aqueous solution. Mix well and react at 30℃ and 1500 rpm for 1 h. After the reaction is complete, add 200 μL of 1M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH=7.0) and continue the reaction for 30 min. Then, add TFA to 1% (V / V) to acidify the solution, centrifuge at high speed to remove the generated bubbles. Finally, remove the salt in the sample with HLB, measure the peptide concentration with nanodrop, and lyophilize the sample in 100 μg tubes.

[0056] (4) 100 μg of peptide was reconstituted in 50 μL of 200 mM sodium hydroxide aqueous solution, and 0.5 M 1,2-cyclohexanedione was added to a final concentration of 20 mM. The reaction was carried out at 37 °C for 40 min. After the reaction was completed, 0.4 M hydrochloric acid was added to adjust the pH of the solution to near neutral. Then, 125 μL of 25 mM triethylamine carbonate buffer (pH 10.2), 50 μL of 100 mM sodium chloride solution, 40 μL of agarose microspheres modified with m-aminophenylboronic acid (Sigma, catalog number A8312) and 1.54 μL of 40% acetone aldehyde solution were added. The reaction was carried out at 15 °C and 1500 rpm for 1 h. Before adding the agarose microspheres, they were washed according to the process and conditions described in step (2) of Example 1. After the reaction, the suspension was transferred to a homemade tip, filtered to remove the solution, and then the microspheres were washed with 50 μL of 10 mM triethylamine carbonate buffer (pH 10.0), repeated 4 times. Finally, the enriched peptides were eluted with 250 μL of 0.1% formic acid aqueous solution, 50 μL each time, with an interval of 5 min between each operation; after elution, all eluents were combined and lyophilized for storage.

[0057] (5) The lyophilized peptide was reconstituted in 60 μL of 80% phosphate buffer (5 mM potassium dihydrogen phosphate, pH = 2.7) / 20% acetonitrile (V / V). 5 μL of strong cation exchange chromatography packing material was added to a self-made tip column, and the column was washed three times with 70% ammonia solution (1% ammonia by mass) / 30% acetonitrile (V / V), with a solution volume of 20 μL each time. The column was then washed six times with 80% phosphate buffer (5 mM potassium dihydrogen phosphate, pH = 2.7) / 20% acetonitrile (V / V), with a solution volume of 20 μL each time. Subsequently, 60 μL of the peptide solution was loaded in three fractions, and the column was washed again with 15 μL of 70% trifluoroacetic acid solution (0.01% trifluoroacetic acid by volume) / 30% acetonitrile (V / V). Finally, the peptides were eluted with 20 μL of 70% ammonia solution (1% ammonia by mass) / 30% acetonitrile (v / v), and the elution was repeated 3 times. All eluents were combined and placed at room temperature for 1 h, and then lyophilized for storage.

[0058] (6) The above experiment was repeated three times. The lyophilized samples were reconstituted in 15 μL of 0.1% formic acid aqueous solution, and the samples were detected by LC-MS. The obtained mass spectrometry files were retrieved using the proteomics analysis software Maxquant. An average of 273 arginine dimethylation sites were identified by single-needle mass spectrometry analysis. The experiment was repeated six times, with two samples combined into one sample. An average of 313 arginine dimethylation sites were identified by single-needle mass spectrometry analysis. In this case, antibody enrichment methods were also used to enrich arginine dimethylated peptides. The antibody enrichment kits developed by Cell Signaling Technology for asymmetric dimethylated arginine (aDMA) and symmetric dimethylated arginine (sDMA) (catalog numbers 13474 and 13563, respectively) were used, following the methods described in the kit instructions. Figure 4 As shown, compared to antibody methods, this method can identify more arginine dimethylation sites. Furthermore, this method is particularly adept at identifying arginine dimethylation sites on the RG and RGG sequences, doubling the number identified compared to antibody methods.

Claims

1. A method for enriching arginine dimethylated peptide based on boron affinity chromatography, characterized in that: a diketone compound is used to selectively react with the unmodified and single-methyl-modified arginine side chain guanidino group of protein enzymatic peptides or peptide mixtures, thereby reducing the chemical reactivity of the two types of guanidino groups; then a vicinal dicarbonyl compound is used to specifically modify a cis vicinal diol group on the arginine side chain guanidino group modified with two methyl groups, and at the same time a chromatographic material modified with a boronic acid group is used to capture the cis vicinal diol group, thereby achieving specific enrichment of arginine dimethylated peptides; the diketone compound is 1,2-cyclohexanedione; the vicinal dicarbonyl compound is methylglyoxal; and the chromatographic material of the boronic acid group is agarose chromatographic filler of m-aminophenylboronic acid. The process for obtaining protein enzymatic peptides includes protein sample enzymolysis to obtain a protein enzymolysis solution, addition of an amino blocking reagent for reaction, desalting, and drying to obtain a peptide mixture. The specific process is as follows: (a) 1 mg of a protein sample is dispersed in 0.2-2 mL of a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer aqueous solution or a triethylamine carbonate buffer aqueous solution with a molar concentration of 10-200 mM and a pH of 7.0-9.0; a protease is added to enzymolyze the dispersed protein sample in the buffer to obtain a protein enzymolysis solution; (b) an amino blocking reagent is added to the protein enzymolysis solution for reaction; after the reaction is completed, the sample is subjected to desalting, and then vacuum freeze-drying is used to freeze-dry the sample to obtain a peptide mixture A.

2. The method of claim 1, wherein, The protein sample source in step (a) includes one or more than two of animal cells, plant cells, animal tissues, plant tissues, or other sources; The protease in step (a) is one or more than two of trypsin, lysyl endopeptidase, clostripain, Staphylococcus aureus V8 protease, pepsin, and carboxypeptidase B, and the enzymolysis conditions in step (a) are as follows: the mass ratio of the protease to the protein sample is 1:10-1:100, the enzymolysis time is 1-24 h, the enzymolysis temperature of carboxypeptidase B is 37-55 ℃, and the enzymolysis temperature of other proteases is 20-37 ℃. The protein sample source in step (a) includes a protein sample from a laboratory cultivable animal cancer cell source; Trypsin is used for enzymolysis first, clostripain is used for enzymolysis second, and carboxypeptidase B is used for further enzymolysis last, and the mass ratio of the three proteases to the protein sample is all in the range of 1:20-1:100; the reaction temperature of the first two steps of enzymolysis is both 35-37 ℃, and the reaction time is both 14-16 h; the reaction temperature of the third step of enzymolysis is 47-50 ℃, and the reaction time is 1.5-2 h.

3. The method of claim 2, wherein, ​ ​ 4. The method of claim 3, wherein, ​ ​ 5. The method of claim 2, wherein, The amino blocking reagent in step (b) is a combination of sodium cyanoborohydride and formaldehyde, and the reaction conditions in step (b) are as follows: 20-200 μL of 0.3-1.0 M sodium cyanoborohydride and 20-200 μL of 1-10% formaldehyde aqueous solution are added to the proteolytic solution obtained in step (a), the reaction temperature is 20-40 ℃, and the reaction time is 0.2-2 h.

6. The method of claim 2, wherein, The desalting in step (b) is performed by using a C18 microsphere packed solid phase microextraction column or a solid phase microextraction column packed with HLB filler developed by Waters Company.

7. The method of any one of claims 1-6, wherein, The steps of the method are as follows: (1) 100 μg of the peptide segment mixture A is resuspended in 50-100 μL of 100-400 mM sodium hydroxide aqueous solution, and a diketone compound is added for reaction; after the reaction is completed, 0.05-12 M hydrochloric acid aqueous solution is used to adjust the pH of the solution to about 5-9, to obtain a peptide segment mixture B; (2) 100-250 μL of 25-50 mM triethylamine carbonate buffer solution is added to the peptide segment mixture B, the pH of the buffer solution is 10-11, 0.1-1 M sodium chloride aqueous solution is further added to a final concentration of 40-100 mM, and then o-dicarbonyl compounds and 5-80 μL of chromatographic material modified with a boronic acid group are added, and the mixture is placed at 10-25 ℃ for 0.5-2 h; after the reaction is completed, the chromatographic material is collected by filtration, and the chromatographic material is washed 3-6 times with 5-25 mM triethylamine carbonate buffer solution, each time using 20-100 μL of the solution, and the pH of the buffer solution is 9.6-10.2; finally, an acidic aqueous solution is used to elute the peptide segments adsorbed on the chromatographic filler, and then the eluate is freeze-dried by a vacuum freeze-drying method to obtain crude arginine dimethylated peptide segments; (3) 5-20 μL of a strong cation exchange chromatographic filler is prepared into a solid phase microextraction column, the column body is washed 2-6 times with elution buffer, each time using 5-40 μL of the solution; the column body is further washed 2-6 times with sample loading buffer, each time using 5-40 μL of the solution; the crude arginine dimethylated peptide segments are resuspended in 40-100 μL of sample loading buffer, and then loaded into the solid phase microextraction column, and the column body is washed 2-6 times with impurity removal buffer, each time using 5-20 μL of the solution; finally, the column body is eluted with 40-100 μL of elution buffer to obtain elution products; The elution products are incubated at 20-37 ℃ for 0.4-2 h, and then freeze-dried to obtain arginine dimethylated peptide segments.

8. The method of claim 7, wherein, The reaction conditions in step (1) are as follows: 1,2-cyclohexanedione is added to the resuspended peptide segment mixture A to a final concentration of 10-40 mM, the reaction temperature is 30-40 ℃, and the reaction time is 0.5-2 h.

9. The method of claim 8, wherein, The specific conditions of the reaction in step (2) are as follows: 1-5 μL of 10-40% (mass concentration) methylglyoxal aqueous solution is added into the reaction system.

10. The method of claim 7, wherein, The acid aqueous solution in step (2) includes one or more than two of 0.01-0.2% (volume concentration) formic acid aqueous solution, 0.01-0.2% (volume concentration) trifluoroacetic acid aqueous solution, 1-10 mM hydrochloric acid aqueous solution, 1-10 mM sulfuric acid aqueous solution or 1-10 mM nitric acid aqueous solution, and the volume of the acid aqueous solution used for eluting the peptide segment is 250-500 μL.

11. The method of claim 7, wherein, The loading buffer in step (3) is a solution prepared by mixing an organic solvent and a buffer salt aqueous solution in a certain proportion; the organic solvent includes one or more than two reagents combined from methanol, ethanol and acetonitrile, and the volume concentration of the organic solvent in the loading buffer is 10-30%; the buffer salt in the buffer salt aqueous solution includes one or more than two of potassium dihydrogen phosphate and sodium dihydrogen phosphate, and the molar concentration of the buffer salt in the aqueous solution is 5-15 mM, and the pH of the buffer salt aqueous solution is 2.0-3.0; The impurity-removing buffer in step (3) is a solution prepared by mixing an organic solvent and an acid aqueous solution in a certain proportion; the organic solvent includes one or more than two reagents combined from methanol, ethanol and acetonitrile, and the volume concentration of the organic solvent in the impurity-removing buffer is 10-30%; the acid in the acid aqueous solution includes trifluoroacetic acid, acetic acid or formic acid, and the volume concentration of the acid in the acid aqueous solution is 0.005-0.1%; The elution buffer in step (3) is a solution prepared by mixing an organic solvent and a basic aqueous solution in a certain proportion; the organic solvent includes one or more than two reagents combined from methanol, ethanol and acetonitrile, and the volume concentration of the organic solvent in the elution buffer is 10-30%; the base in the basic aqueous solution includes ammonia, monomethylamine, dimethylamine, trimethylamine or triethylamine, and the mass concentration of the base in the basic aqueous solution is 0.5-2%.

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