Method for weakening drug resistance of tumor cells
阅读说明：本技术 一种减弱肿瘤细胞耐药性的方法 (Method for weakening drug resistance of tumor cells ) 是由 叶明亮 刘震 王麒 王科云 于 2019-11-28 设计创作，主要内容包括：本发明涉及一种细胞培养条件下稳定同位素标记技术(Stable isotope labeling with amino acids in cell culture,SILAC)结合固相萃取-强阳离子交换树脂分离敏感和耐药细胞甲基化位点的方法。细胞在含有重标甲硫氨酸的培养基中培养,利用胰蛋白酶Trypsin和赖氨酸酶Lys-C酶解,再使用精氨酸酶Arg-C进行酶解,SCX分级蛋白酶解物的肽段,最后将洗脱下来的肽段进行LC-MS/MS分析。通过labelfree定量可以得出差异的赖氨酸和精氨酸甲基化位点,将这些甲基化位点进行motif分析后发现敏感和耐药细胞的赖氨酸甲基转移酶偏好性明显不同,比较获得耐药和敏感细胞中对应甲基化修饰位点表达量的比值,将绝对值最大的甲基位点所对应的甲基转移酶进行含量调整,从而减弱肿瘤细胞的耐药性。(The invention relates to a method for separating sensitive and drug-resistant cell methylation sites by combining Stable isotope labeling technology (SILAC) with solid phase extraction-strong cation exchange resin under cell culture conditions. Culturing cells in a culture medium containing the re-standard methionine, performing enzymolysis by Trypsin Trypsin and lysine Lys-C, performing enzymolysis by arginase Arg-C, classifying peptide fragments of the protein zymolyte by SCX, and finally performing LC-MS/MS analysis on the eluted peptide fragments. Different lysine and arginine methylation sites can be obtained through labelfree quantification, the methylation sites are analyzed for motif, the lysine methyltransferases of sensitive cells and drug-resistant cells have obviously different preferences, the ratio of the expression amounts of the corresponding methylation modification sites in the drug-resistant cells and the sensitive cells is obtained through comparison, and the content of the methyltransferase corresponding to the methyl site with the maximum absolute value is adjusted, so that the drug resistance of tumor cells is weakened.)
1. A method of reducing resistance to tumor cells, comprising:
respectively culturing sensitive and drug-resistant tumor cells in a culture medium containing heavy-duty methionine, taking out the cultured cells, respectively extracting proteins, respectively performing enzymolysis on the proteins by Trypsin and lysase Lys-C, respectively performing enzymolysis by arginase Arg-C, respectively performing solid phase extraction on protein zymolytes according to the ionic interaction strength to separate peptide segments with positive charges, performing LC-MS/MS analysis on the mixture of lysine and arginine methylated peptide segments of the sensitive and drug-resistant cells by using a filler which is strong cation exchange resin, quantitatively obtaining different lysine and arginine methylated sites by label free, performing motif analysis on the methylated sites to find that the lysine methyltransferases of the sensitive and drug-resistant cells are obviously different, and comparing to obtain the ratio of the expression quantity of the corresponding methylated modified sites in the drug-resistant and sensitive cells, sequencing the absolute value of the difference between the ratio and 1 from large to small, and taking lysine methyltransferases arranged at the corresponding sites of the first 1-20 (preferably 1-10, most preferably 1-5, and more preferably 1-2) from the end with large absolute value for content adjustment, wherein the lysine methyltransferases at the corresponding sites are required to be reduced in the cell on the side more than 1, and the lysine methyltransferases at the corresponding sites are required to be increased in the cell on the side less than 1; the drug resistance of the drug-resistant cells is weakened.
2. The method of claim 1, wherein:
the specific steps for enriching lysine and arginine methylation sites of sensitive and drug-resistant cells are as follows:
(1) respectively culturing sensitive and drug-resistant tumor cells in a culture medium containing re-labeled methionine for 1-10 generations, respectively dissolving the extracted protein in a buffer solution containing 1-10M Urea and 10-100mM Tris, adding Dithiothreitol (DTT) with the final concentration of 10-100mM in a water bath at 10-100 ℃ for 1-10h, then adding Iodoacetamide (IAA) with the final concentration of 10-100mM, and carrying out light-shielding reaction at 10-100 ℃ for 10-100 min;
(2) carrying out ultrafiltration centrifugation on the sample obtained after the step (1) by using an ultrafiltration tube with the molecular weight cutoff of 3-30K under the centrifugal force of 1000-20000g, adding an enzymolysis buffer solution into the trapped sample, then respectively adding Trypsin Trypsin and lysine Lys-C with the protein mass ratio of 1/100-1/25, and carrying out enzymolysis for 12-24h in water bath at 37 ℃ to obtain a peptide fragment solution;
(3) carrying out ultrafiltration centrifugation on the sample obtained after the step (2) by using an ultrafiltration tube with the molecular weight cutoff of 3-30K under the centrifugal force of 1000-;
(4) adding trifluoroacetic acid into the peptide fragment solution obtained in the step (3) to adjust the pH value to 2-3, then removing small molecules in the solution by using a C18 solid phase extraction column, and freeze-drying the obtained peptide fragment eluent;
(5) re-dissolving 100-;
(6) adding 100-;
(7) adding 100-;
(8) adding 100-;
(9) adding 100-;
(10) and (3) adding trifluoroacetic acid into the eluates obtained in the step (7), the step (8) and the step (9) to adjust the pH value to 2-3, removing small molecules in the solution by using a C18 solid phase extraction column, and freeze-drying the obtained peptide fragment eluent.
3. The method of claim 2, wherein:
the final concentration of the re-labeled methionine in the step (1) is 10-30 mg/L.
4. The method of claim 2, wherein:
the enzymolysis buffer solution in the step (2) is 5-100mM of tris (hydroxymethyl) aminomethane, and the pH value is 7-10 aqueous solution.
5. The method of claim 2, wherein:
the activation buffer solution in the step (3) is 5-50mM of tris (hydroxymethyl) aminomethane, the pH value is 7-10,5-50mM of dithiothreitol, 0.2-2mM of ethylenediamine tetraacetic acid and 50-500mM of calcium chloride.
6. The method of claim 2, wherein:
the loading buffer solution in the steps (5) and (6) is 1-10mM potassium dihydrogen phosphate, the pH value is 1.7-5.7, and the acetonitrile solution of 10-100% (V/V) acetonitrile;
the washing buffer solution 1 in the step (7) is 1-10mM potassium dihydrogen phosphate, 10-100mM potassium chloride, pH value is 1.7-5.7, and acetonitrile solution of 10-100% (V/V) acetonitrile;
the washing buffer solution 2 in the step (8) is 1-10mM potassium dihydrogen phosphate, 10-100mM potassium chloride, pH value is 1.7-5.7, and acetonitrile solution of 10-100% (V/V) acetonitrile;
the elution buffer in steps (6) and (9) is 1-10mM potassium dihydrogen phosphate, 100-1000mM potassium chloride, and acetonitrile solution with pH value of 5-10 and 10-100% (V/V) acetonitrile.
7. The method according to claim 1 or 2, characterized in that: the tumor cell is one of liver cancer, lung cancer, breast cancer, ovarian cancer and the like;
the sensitive tumor cell and the drug-resistant tumor cell are the same tumor cell;
the culture medium is required by corresponding tumor cells.
8. The method according to claim 1 or 2, characterized in that:
taking tumor cells derived from tumor organisms which are taken with a medicament to be detected as standby drug-resistant tumor cells;
taking tumor cells derived from tumor organisms which have not been taken with a medicament to be detected as standby sensitive tumor cells;
culturing standby drug-resistant tumor cells and standby sensitive tumor cells by using the drugs to be detected with the same final concentration to obtain IC50 values of the standby drug-resistant tumor cells and the standby sensitive tumor cells respectively, wherein if the ratio of the IC50 values of the standby drug-resistant tumor cells to the IC50 values of the standby sensitive tumor cells is more than or equal to 10, the standby drug-resistant tumor cells and the standby sensitive tumor cells can be used as the drug-resistant tumor cells and the sensitive tumor cells for experiments;
the medicine to be detected can be a medicine which can be taken by organisms with tumor diseases and can treat tumors, the medicine can be fluorouracil (5-Fu), cisplatin medicines, tamoxifen and the like, and the organisms with the tumor diseases can be white mice and white rabbits.
9. The method according to claim 1 or 2, characterized in that:
the obtained methylated peptide containing lysine and arginine is redissolved in trifluoroacetic acid with a volume concentration of 0.01-10% for RP LC-MS/MS analysis.
10. The method according to claim 1 or 2, characterized in that:
the method can simultaneously enrich monomethylation arginine, dimethylation arginine, monomethylation lysine, dimethylation lysine and trimethylation lysine of sensitive and drug-resistant cells, and the antibody can only enrich corresponding methylation forms.
Technical Field
The invention belongs to the field of tumor cell drug resistance research in the proteomics research direction, and particularly relates to a method for enriching lysine and arginine methylated peptide fragments by combining a stable isotope labeling technology with solid phase extraction-strong cation exchange resin under a cell culture condition.
Background
Cancer is a common malignant tumor and a serious disease which easily causes death of human beings, and some chemotherapeutic drugs can kill cancer cells well, but can easily cause drug resistance of the cancer cells after long-term administration (document 1.Li, X.; Yu, J.; Brock, M.V.; Tao, Q.; Herman, J.G.; Liang, P.; Guo, M., Epigenic sizing of BCL6B inactivates p53 signaling and consumers human hepatocyte resistance 2015,6, (13),11547 and 11560.), so understanding the molecular mechanism of drug resistance of the cancer cells is very critical for identifying new therapeutic targets to weaken the resistance of the cells.
Intracellular signal transduction is often accomplished by different post-translational modifications of proteins, such as phosphorylation and methylation, the basic cellular transduction pathways of phosphorylation being well defined. Although methylation and phosphorylation were found to be synchronized in time, signal transduction pathways for protein methylation have not been well documented. Methylation typically occurs at arginine and lysine residues of proteins. Lysine can form monomethylation, dimethylation and trimethylation with S-adenosylmethionine as a methyl donor by lysine methyltransferase (document 2.Paik, W.K., Paik, D.C., Kim, S., History review: the field of Protein methylation. trends biochem. Sci.2007,32, 146. 152, document 3.Lake, A.N., Bedford, M.T., Protein methylation and DNA repair. Mutat. Res. -Fundam.mol.Mel.Mutagen.2007, 618,91-101, document 4.Smith, B.C., Denu, J.M., Chemical, machinery of Protein methylation and tissue modification, Biophyca. Achysica-Biophyca. 2009, Regulation 2009, 1745). Arginine side chains form monomethylation and dimethylation by Arginine methyltransferase (document 5.Bedford, M.T., Richard, S., Arginine methyl: An empirical regulation of protein function. molecular Cell 2005,18, 263-272), wherein dimethylation is further divided into symmetric dimethylation and asymmetric dimethylation. There is a large body of evidence that protein methylation plays a key role in maintaining intracellular function and cellular physiological and pathological changes. Histone methylation affects physical properties of chromatin, affecting gene expression (literature 6.Zhou, v.w., Goren, a., Bernstein, b.e., Charting histomodifications and the functional organization of mammalian genes. nature Reviews Genetics 2011,12, 7-18.). In addition to histones, methylation of lysine and arginine also occurs in non-histones and plays an important regulatory role (7. Paik, W.K., Paik, D.C., Kim, S., Historial review: the field of protein methylation. trends biochem. Sci.2007,32, 146. 152.), however, how to generate drug-resistant methylation in tumor cells has not been reported.
In order to better understand the state change of protein methylation in tumor cell drug resistance, the invention is based on the combination of the re-labeled methionine mark and the solid phase extraction-strong cation exchange resin to enrich lysine and arginine methylation peptide fragments. By analyzing methylation sites motif of sensitive and drug-resistant cells, the preference of methyl transferase catalyzing the cells is obviously different, the ratio of the expression quantity of corresponding methylation modification sites in the drug-resistant and sensitive cells is obtained by comparison, and the content of the methyl transferase corresponding to the methyl site with the largest absolute value is adjusted, so that the drug resistance of tumor cells is weakened.
Disclosure of Invention
The invention aims to provide a method for enriching lysine and arginine methylation sites of sensitive and drug-resistant tumor cells, which is simple, convenient, low in cost and good in repeatability.
The method provided by the invention is characterized in that a methylated peptide fragment is separated from a complex sample by utilizing a method of culturing heavy-duty methionine and combining solid phase extraction-strong cation exchange resin, and then lysine and arginine methylation sites of the sample are obtained through mass spectrometry.
The invention adopts the following technical scheme:
cell samples cultured by re-scaling methionine are subjected to enzymolysis by Trypsin and Trypsin Lys-C, and then are subjected to enzymolysis by arginase Arg-C, and then methylated peptide fragments are separated by solid phase extraction-strong cation exchange resin, and finally the eluted peptide fragments are subjected to LC-MS/MS analysis to obtain the identification results of lysine and arginine methylation sites.
The specific steps for enriching lysine and arginine methylation sites of sensitive and drug-resistant cells are as follows:
(1) respectively culturing sensitive and drug-resistant tumor cells in a culture medium containing re-labeled methionine for 1-10 generations, respectively dissolving the extracted protein in a buffer solution containing 1-10M Urea and 10-100mM Tris, adding Dithiothreitol (DTT) with the final concentration of 10-100mM in a water bath at 10-100 ℃ for 1-10h, then adding Iodoacetamide (IAA) with the final concentration of 10-100mM, and carrying out light-shielding reaction at 10-100 ℃ for 10-100 min;
(2) carrying out ultrafiltration centrifugation on the sample obtained after the step (1) by using an ultrafiltration tube with the molecular weight cutoff of 3-30K under the centrifugal force of 1000-20000g, adding an enzymolysis buffer solution into the trapped sample, then respectively adding Trypsin Trypsin and lysine Lys-C with the protein mass ratio of 1/100-1/25, and carrying out enzymolysis for 12-24h in water bath at 37 ℃ to obtain a peptide fragment solution;
(3) carrying out ultrafiltration centrifugation on the sample obtained after the step (2) by using an ultrafiltration tube with the molecular weight cutoff of 3-30K under the centrifugal force of 1000-;
(4) adding trifluoroacetic acid into the peptide fragment solution obtained in the step (3) to adjust the pH value to 2-3, then removing small molecules in the solution by using a C18 solid phase extraction column, and freeze-drying the obtained peptide fragment eluent;
(5) re-dissolving 100-;
(6) adding 100-;
(7) adding 100-;
(8) adding 100-;
(9) adding 100-;
(10) and (3) adding trifluoroacetic acid into the eluates obtained in the step (7), the step (8) and the step (9) to adjust the pH value to 2-3, removing small molecules in the solution by using a C18 solid phase extraction column, and freeze-drying the obtained peptide fragment eluent.
The final concentration of the re-labeled methionine in the step (1) is 10-30 mg/L.
The enzymolysis buffer solution in the step (2) is 5-100mM of tris (hydroxymethyl) aminomethane, and the pH value is 7-10 aqueous solution.
The activation buffer solution in the step (3) is 5-50mM Tris (hydroxymethyl) aminomethane with the pH value of 7-10,5-50mM dithiothreitol, 0.2-2mM ethylene diamine tetraacetic acid and 50-500mM calcium chloride.
In the steps (5) and (6), the loading buffer solution is 1-10mM potassium dihydrogen phosphate, the pH value is 1.7-5.7, and the acetonitrile solution of 10-100% (V/V) acetonitrile.
The washing solution 1 in the step (7) is 1-10mM potassium dihydrogen phosphate, 10-100mM potassium chloride, pH 1.7-5.7, 10-100% (V/V) acetonitrile solution in acetonitrile.
The washing solution 2 in the step (8) is 1-10mM potassium dihydrogen phosphate, 10-100mM potassium chloride, pH 1.7-5.7, 10-100% (V/V) acetonitrile solution in acetonitrile.
The elution solution in the step (9) is 1-10mM potassium dihydrogen phosphate, 100-1000mM potassium chloride, and acetonitrile solution with pH value of 5-10, 10-100% (V/V) acetonitrile.
The tumor cell is one of liver cancer, lung cancer, breast cancer, ovarian cancer and the like;
the sensitive tumor cell and the drug-resistant tumor cell are the same tumor cell;
the culture medium is required by corresponding tumor cells.
Taking tumor cells derived from tumor organisms which are taken with a medicament to be detected as standby drug-resistant tumor cells;
taking tumor cells derived from tumor organisms which have not been taken with a medicament to be detected as standby sensitive tumor cells;
culturing standby drug-resistant tumor cells and standby sensitive tumor cells by using the drugs to be detected with the same final concentration to obtain IC50 values of the standby drug-resistant tumor cells and the standby sensitive tumor cells respectively, wherein if the ratio of the IC50 values of the standby drug-resistant tumor cells to the IC50 values of the standby sensitive tumor cells is more than or equal to 10, the standby drug-resistant tumor cells and the standby sensitive tumor cells can be used as the drug-resistant tumor cells and the sensitive tumor cells for experiments;
the medicine to be detected can be a medicine which can be taken by organisms with tumor diseases and can treat tumors, the medicine can be fluorouracil (5-Fu), cisplatin medicines, tamoxifen and the like, and the organisms with the tumor diseases can be white mice and white rabbits.
The lysine and arginine methylated peptide fragments obtained in the above steps (7), (8) and (9) were redissolved in trifluoroacetic acid at a concentration of 0.1% by volume for RPLC-MS/MS analysis.
The method has the advantages of low cost, simple operation and good repeatability, and can be used for methylation proteome analysis of sensitive and drug-resistant cells.
The methylated peptide fragment enrichment material used was commercial polar MC30-SP SCX beads (Sepax).
According to the methylated peptide enrichment method, lysine and arginine methylated peptide in protein enzymatic hydrolysate are separated by strong cation exchange resin according to the ion exchange strength of the lysine and arginine methylated peptide.
The invention has the advantages that:
the enrichment method has the advantages of low cost, easy operation and good reproducibility. The invention utilizes the combination of solid phase extraction-strong cation exchange resin under the condition of culturing the re-labeled methionine for the drug resistance analysis of tumor cells and the RPLC-MS/MS analysis with high resolution, effectively enriches lysine and arginine methylation sites, can obtain different lysine and arginine methylation sites through label free quantification, finds that the lysine methyltransferases of sensitive cells and drug-resistant cells have obviously different preferences after the methylation sites are subjected to motif analysis, obtains the ratio of the expression quantity of corresponding methylation modification sites in the drug-resistant cells and the sensitive cells by comparison, and adjusts the content of the methyltransferase corresponding to the methyl site with the maximum absolute value, thereby weakening the drug resistance of the tumor cells.
Drawings
FIG. 1 is a flow chart of the method for the proteome analysis of the methylation of sensitive and resistant tumor cells. Culturing sensitive and drug-resistant cells in a culture medium containing heavy-duty methionine, carrying out enzymolysis on protein by using a plurality of enzymes, separating peptide fragments after enzymolysis by using solid phase extraction-strong cation exchange resin, carrying out Q active HF analysis on the eluted peptide fragments, and then quantifying by using a label free method.
FIG. 2 shows the analysis method of the methylation proteome of sensitive and drug-resistant cells, which comprises injecting DEPC water into 50 tumor-bearing mice at multiple points every week, injecting 5-Fu (3mg/kg) into 50 tumor-bearing mice at multiple points every week, adding 1mg/kg of drug every week for 10 weeks, and recording the volume of the tumor. ,
FIG. 3 is a proteome analysis method for the methylation of sensitive and resistant cells, tumor cells of the control and drug groups were injected with 5-Fu at 0mg/kg, 1mg/kg, 3mg/kg, 9mg/kg and 18mg/kg of the drug, and tumor volume sizes were recorded after one week and repeated 3 times at each concentration.
FIG. 4 is a graph of the sensitive and drug-resistant cell methylation proteome analysis method for the quantitative analysis of data in tumor cell methylation proteomics of control and drug-administered groups, wherein the results are quantitative and significantly changed methylation forms and methylated proteins.
FIG. 5 is the sensitive and drug resistant cell methylation proteomic analysis method for quantitative analysis data of tumor cell methylation proteomics of control and drug groups, (a) the motif of the lysine methylation site up-regulated, (b) the motif of the arginine methylation site up-regulated, (c) the motif of the lysine methylation site down-regulated, (d) the motif of the arginine methylation site down-regulated.
FIG. 6 is the analysis method of the methylation proteome of the sensitive and resistant cells for weakening the growth data of the tumor cells in the drug group, the tumor body of the drug group is injected with DEPC water 50 μ L at multiple points + sodium chloride injection 0.9% 200 μ L in the abdominal cavity, the tumor body is injected with DEPC water 50 μ L at multiple points + 5-Fu (3mg/kg) in the abdominal cavity, the tumor body is injected with siRNA 500pmol + sodium chloride injection 0.9% 200 μ L in the abdominal cavity, the tumor body is injected with siRNA 500pmol + 5-Fu (3mg/kg) at multiple points + the abdominal cavity, once per week, 4 times in total, and the tumor volume size is recorded.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Examples
The hepatoma cell line Bel-7402 is cultured in 1640 culture medium containing 10% fetal calf serum and cultured in an incubator at 5% and 37 ℃. Preparing single cell suspension from logarithmic growth phase cells, and adjusting cell density to 1 х 108one/mL. The single cell suspension was injected subcutaneously into the right back of nude mice in a dose of 200. mu.L each with a 1mL syringe. Strictly according to the aseptic operating principle. Observing the inoculation part every other day after the inoculation is finished, measuring the longest warp and the shortest warp of the tumor by using a vernier caliper, and calculating the tumor volume according to the following formula: v (mm)3)＝πabc/6。
When the volume of the transplanted tumor is more than 10mm3At the time, 100 tumor-bearing mice were randomly divided into 2 groups, i.e., a control group and a drug group, and each group had 50 mice. Control group: injecting DEPC water into tumor body at multiple points; the medicine group is as follows: tumor was injected with 5-Fu (3mg/kg) at multiple points 1 time per week, with 1mg/kg drug added per week for a total of 10 times.
Before each injection, the longest meridian a, the shortest meridian b and the height c of the tumor are measured by a vernier caliper, the tumor volume is calculated according to a formula, and the tumor volume is recorded.
Taking 15 tumor-bearing mice of a control group and 15 drug groups, injecting 5-Fu of 0mg/kg, 1mg/kg, 3mg/kg, 9mg/kg and 18mg/kg into tumor bodies of the tumor-bearing mice for one week, repeating each concentration for 3 times, calculating the tumor volume according to a formula, and calculating an IC50 value.
After determining that the tumor body of the tumor-bearing mouse has drug resistance, feeding food containing the re-labeled methionine for 2 weeks, injecting 5-Fu of 3mg/kg once a week into a drug group to maintain the drug resistance, killing the tumor-bearing mouse, taking tumor tissues, and extracting protein.
(1) Dissolving 1mg of protein of control group and drug group in buffer solution of 8M Urea and 50mM Tris, adding 20mM DTT, carrying out water bath at 37 ℃ for 2h, then adding 40mM IAA, and carrying out light-shielding reaction at 25 ℃ for 40 min;
(2) carrying out ultrafiltration centrifugation by using a 10K ultrafiltration tube at 14000g, adding 10mM of tris (hydroxymethyl) aminomethane into an intercepted sample, wherein the pH value is 8.0, then respectively adding Trypsin and lysase Lys-C with the mass ratio of the Trypsin to the protein being 1/100, and carrying out enzymolysis for 16h in a water bath at 37 ℃ to obtain a peptide fragment solution;
(3) carrying out ultrafiltration centrifugation by using a 10K ultrafiltration tube at 14000g, adding 5mM tris (hydroxymethyl) aminomethane with the final concentration of 5mM, the pH value of 7.6,5mM dithiothreitol, 0.2mM ethylene diamine tetraacetic acid and 5mM calcium chloride buffer solution into an intercepted sample, then adding arginase Arg-C with the mass ratio of protein of 1/100 into the intercepted sample, and carrying out enzymolysis for 16h in water bath at 37 ℃ to obtain a peptide fragment solution;
(4) adding trifluoroacetic acid into the peptide fragment solution, adjusting the pH value to 2-3, removing small molecules in the solution by using a C18 solid phase extraction column, and freeze-drying the obtained peptide fragment eluent;
(5) add 200. mu.L of SCX material to a 2.5ml SPE cartridge, wash 3 times with 300. mu.L elution buffer (5mM potassium dihydrogen phosphate, 500mM potassium chloride, pH 7.0, 30% acetonitrile), then equilibrate 3 times with 200. mu.L loading buffer (5mM potassium dihydrogen phosphate, pH 2.7, 30% acetonitrile);
(6) re-dissolving 1000 μ g of the enzymolysis peptide segment in 900 μ L of the sample loading buffer solution, and adding the obtained solution into an SPE column;
(7) washing SPE column with 300 μ L of washing buffer 1(5mM potassium dihydrogen phosphate, 30mM potassium chloride, pH 2.7, 30% acetonitrile) for 5 times, and collecting the washing solution;
(8) washing SPE column with 300 μ L of washing buffer 2(5mM potassium dihydrogen phosphate, 40mM potassium chloride, pH 2.7, 30% acetonitrile) for 3 times, and collecting the washing solution;
(9) washing SPE column with 300 μ L of elution buffer solution for 3 times, and collecting eluate;
(10) adding trifluoroacetic acid into the eluate to adjust pH to 2-3, removing small molecules in the solution with C18 solid phase extraction column, and freeze drying the obtained peptide fragment eluate;
(11) the obtained lysine and arginine methylated peptide fragments were redissolved in 0.1% (v/v) trifluoroacetic acid for RP LC-MS/MS analysis.
(12) And (2) quantifying the label free of data collected by mass spectrometry by using a Maxquant search engine, processing the quantitative result by perseus software to obtain different methylation sites, comparing to obtain the ratio of the expression quantity of the corresponding methylation modification sites in the drug-resistant and sensitive cells, sorting the ratio and the absolute value of 1 difference from large to small, taking lysine methyltransferases ranked at the corresponding sites in the front 1 from one end with a large absolute value for content adjustment, wherein the methylation site with the largest absolute value is a dimethylation site di-K561 of HSPA8 (the change multiple is 5.12, and the p value is 0.002), and the methyltransferases are METTL 21A.
(13) The expression of METTL21A in the medicine group is reduced by utilizing siRNA technology, and the specific experimental design is as follows: injecting 50 mu L of DEPC water into the tumor body at multiple points and injecting 200 mu L of 0.9 percent sodium chloride injection into the abdominal cavity; 5-Fu group: injecting DEPC water 50 μ L + intraperitoneally 5-Fu (3mg/kg) at multiple points of tumor; group of sirnas: tumor body multi-point injection siRNA 500pmol + intraperitoneal injection 0.9% sodium chloride injection 200 mu L; siRNA in combination with 5-Fu group: tumor volume was recorded by multipoint siRNA 500pmol + intraperitoneal 5-Fu injection (3mg/kg), once a week for a total of 4 times.
FIG. 2 shows the change of tumor volume during the drug-adding process of tumor cells, and it can be seen that the tumor volume of the control group gradually increases with time, whereas the tumor volume of the drug-added group gradually decreases with time at the beginning, the tumor volume is minimal at the fifth week, and then the tumor volume gradually increases with time, indicating that the drug-added group tumor cells have drug resistance.
FIG. 3 shows the IC50 values of tumor cells of the control group and the drug group, the IC50 value of the control group is 2.069mg/kg, the IC50 value of the drug group is 61.83mg/kg, and the IC50 value of the drug group is 29.9 times of that of the control group, so that the drug group can be used for the next experiment.
FIG. 4 shows the results of quantitative analysis of methylation sites, and it can be seen that a total of amounts of methylation patterns is 231, 89 are statistically significant, and 15 methylation patterns are found to be up-regulated and include 9 arginine methylation patterns and 6 lysine methylation patterns, 74 methylation patterns are down-regulated and include 42 arginine methylation patterns and 32 lysine methylation patterns, and the remaining 142 methylation patterns are unchanged, and these differential methylation patterns may play an important role in tumor cell resistance.
FIG. 5 shows the results of a motif analysis of methylation sites, from which it can be seen that the sequence characteristics of the up-regulated lysine methylated form are significantly different from those of the down-regulated methylated form, and further we have found that lysine methyltransferases in tumor cells in a drug group prefer a lysine with arginine in the ortho-methylation position, which suggests that methyltransferases of the type that prefer methylation may be associated with drug resistance in tumor cells.
FIG. 6 shows the results of the tumor cell growth volume curve, from which it can be seen that the tumor volume change trends of the control group, the 5-Fu group and the siRNA are almost consistent with the increase of time, whereas the tumor cell growth of the siRNA and 5-Fu group is obviously inhibited.
In summary, the invention is a method for separating methylation sites of sensitive and drug-resistant cells by combining a stable isotope labeling technology with solid phase extraction-strong cation exchange resin under cell culture conditions. The method is easy to operate, low in cost and good in reproducibility, can simultaneously enrich lysine and arginine methylation sites of sensitive and drug-resistant cells, can obtain different lysine and arginine methylation sites through labelfree quantification, and adjusts the content of methyltransferase corresponding to the different methylation sites, thereby weakening the drug resistance of tumor cells.