CN113155577A - Preparation method for identifying specific protein acetylation modified sample - Google Patents
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Abstract
The invention belongs to the technical field of biotechnology, and particularly discloses a preparation method for identifying a specific protein acetylation modified sample. The preparation method comprises the following steps: 1) selecting protein A with acetylation modification and acetyltransferase B; 2) preparing protein samples of co-transfected plasmid A and plasmid B and protein samples of singly transfected plasmid A; 3) selecting a protein sample of co-transfected plasmid A and plasmid B and a protein sample of single transfected plasmid A to complete immunoblotting analysis; 4) and (3) running SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) glue on the rest protein samples of the cotransfection plasmid A and the plasmid B, incubating overnight in Coomassie brilliant blue staining solution, decoloring, cutting a target strip, and putting the cut target strip into a centrifugal tube to obtain the acetylation modified sample for identifying the specific protein. The protein sample prepared by the invention can obviously enhance the success rate of mass spectrum identification of specific protein acetylation modification sites.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of biology, in particular to a preparation method for identifying a specific protein acetylation modified sample.
[ background of the invention ]
Post-translational modification (PTM) of a protein refers to chemical modification of a protein after translation, which participates in almost all normal life processes of a cell and plays an important role in regulation. PTM, as a means of protein function regulation, is critical to the structure and function of proteins. Studies have shown that 50% to 90% of proteins in humans undergo post-translational modifications. More than 400 post-translational modifications have been identified, with common modifications including methylation, phosphorylation, ubiquitination, acetylation, glycosylation, SUMO, nitrosylation, and oxidation, among others. Therefore, PTM has become an extremely important field of protein research in the world. Among them, protein acetylation is a ubiquitous, reversible and highly regulated post-translational modification of proteins, occurring mainly at the epsilon-NH 2 position of protein lysine residues. Acetylation is regulated by both acetylases and deacetylases and is involved in almost all biological processes such as transcription, stress, metabolism, and protein synthesis and degradation.
The detection of protein acetylation modification is different from phosphorylation, and phosphorylation can be studied by a stable and effective means such as an in-situ phosphorylation marker and a sensitive phosphorylation specific antibody, and is relatively easy to detect. However, the means and methods for detecting acetylation of proteins are very limited, and these technical difficulties limit the research and development of protein acetylation.
The main methods for detecting protein lysine acetylation in the prior art comprise: (1) enriching acetylated peptide fragments by using lysine acetylated specific antibodies, and detecting by using a liquid chromatography-mass spectrometry (HPLC/MS) method; (2) the identification of protein acetylation is completed by stable isotope labeling with amino acids in cell culture (SILAC) technology and high-resolution and high-sensitivity electric field orbit trap cyclotron resonance (LTQ Orbitrap) mass spectrometer. However, all of the above methods are based on studies on acetylation modification of lysine at the proteomic level, and cannot be used for detecting acetylation modification of a specific protein.
[ summary of the invention ]
In view of the above, there is a need to provide a preparation method for identifying specific protein acetylation modified samples, and the protein samples prepared by the preparation method of the present invention can significantly enhance the success rate of mass spectrometry for identifying specific protein acetylation modified sites.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method for identifying a specific protein acetylation modified sample, comprising the following steps:
1) selecting protein A with acetylation modification through an endogenous experiment, and determining acetyltransferase B of the protein A through an exogenous experiment;
2) preparation of protein samples of co-transfected plasmid a and plasmid B and protein samples of transfected plasmid a alone:
(1) respectively inoculating 293T cells in at least 20 cell culture dishes, and after the cell density reaches 78-82%, singly transfecting the plasmid A into cells in one cell culture dish by using a transfection reagent, and simultaneously co-transfecting the plasmid A and the plasmid B into the cells in the rest cell culture dishes by using the transfection reagent;
(2) placing the transfected cell culture dish into an incubator, culturing for 6 hours at constant temperature and constant carbon dioxide concentration, removing the culture solution containing the transfection reagent-DNA compound, and replacing a new culture solution to continuously culture for 48 hours;
(3) collecting the cultured cells in a first centrifuge tube, centrifuging cell precipitates, and adding RIPA lysate into the first centrifuge tube;
(4) placing the first centrifuge tube on a vertical rotary shaking table for cracking for 1h, centrifuging, transferring cell supernatant obtained by centrifuging into a second centrifuge tube, adding an antibody into the second centrifuge tube, and incubating on the vertical rotary shaking table overnight;
(5) adding agarose beads to the second centrifuge tube the next day, and then continuing to incubate overnight on a vertical rotary shaker;
(6) centrifuging the antigen-antibody-agarose bead compound in the second centrifugal tube on the third day, washing with cold RIPA lysate for 3 times, and sucking dry liquid after the last washing;
(7) adding a sample buffer solution with twice concentration into the obtained compound, boiling for 5-8min at 100 ℃, and then performing instantaneous centrifugation to obtain a protein sample of a single transfection plasmid A and at least 19 protein samples of co-transfection plasmids A and B;
3) randomly extracting 1 protein sample from the protein samples of the co-transfected plasmid A and the plasmid B, and completing immunoblotting analysis together with the protein sample of the single transfected plasmid A;
4) running SDS-PAGE gel on the protein samples of the rest cotransfection plasmids A and B, incubating in Coomassie brilliant blue staining solution overnight, decoloring the gel on a shaking table the next day until the background of the gel is white and the protein band, especially the target band, in the lane is blue, and stopping decoloring; and cutting a target strip according to the molecular weight of the A, putting the target strip into a third centrifuge tube, and marking to obtain the sample for identifying the specific protein acetylation modification.
Further, in the step 2) (1), the amount of the 293T cells inoculated is 5.2X106Per cell culture dish.
Further, in (1) of step 2), the transfection reagent is an EZ Trans transfection reagent.
Further, in the step 2) (2), the constant temperature is 37 ℃, and the concentration of the carbon dioxide is 5%.
Further, in step 2) (1), after the cell density reaches 80%, plasmid A alone transfects cells of one cell culture dish with a transfection reagent, and simultaneously, plasmid A and plasmid B co-transfect cells of the remaining cell culture dishes with the transfection reagent.
Further, in step 1), the exogenous experiment is: in 293T cells, plasmid A and plasmid B were co-transfected, cells were harvested after 48h, co-immunoprecipitation was performed first, and then immunoblot analysis was performed.
The invention has the following beneficial effects:
firstly, determining that the selected protein has acetylation modification through an endogenous experiment, and further determining that the selected protein has acetylation modification through an exogenous experiment, wherein the accuracy is 100%, and the acetyltransferase of the protein can be determined through an exogenous experiment; secondly, the invention effectively enriches the protein samples which are acetylated and not acetylated through the IP technology; thirdly, before the samples are sent for mass spectrum identification, the acetylation modification of all the samples to be detected is ensured through immunoblot analysis, and the existence of invalid samples is avoided; fourth, protein samples subjected to mass spectrometric identification are more amenable to identification of acetylated modification sites by pooling them such that they have sufficiently high abundance. Through the organic combination of the steps, the protein sample prepared by the preparation method can obviously enhance the success rate of mass spectrum identification of specific protein acetylation modification sites.
[ description of the drawings ]
FIG. 1 is a graph showing the result of the acetylation of endogenous TLR9 induced by the ODN of the present invention (i.e., the result of the endogenous experiment).
Fig. 2 is a graph showing the results of detecting exogenous acetylation modification of TLR9 according to the present invention (i.e., exogenous experimental results).
FIG. 3 is a graph showing the results of acetylation modification detection using immunoblot analysis techniques on randomly sampled protein samples.
FIG. 4 is a Coomassie brilliant blue staining of protein samples co-transfected with HA-A and HA-B after running gel.
Fig. 5-11 are both secondary mass spectra of the TLR9 acetylation site.
The following detailed description will further illustrate the invention in conjunction with the above-described figures.
[ detailed description ] embodiments
In a preferred embodiment of the invention, the identification of the site of acetylation modification of TLR9 is described.
A preparation method for identifying a specific protein acetylation modified sample, comprising the following steps:
1) determining that TLR9 has an acetylated modification by endogenous experiments and that the acetyltransferase CBP of TLR9 is determined by exogenous experiments;
wherein, the endogenous experiment specifically comprises: mouse macrophage Raw264.7 was stimulated at different time points (0min, 15min, 30min) with the same concentration of TLR9 receptor activator ODN (0.5 μ M), and a deacetylase inhibitor (Trichostatin A, TSA) was added to stimulate the above cells based on ODN stimulation for 30min, and then the cells were collected for Immunoprecipitation (IP) and immunoblot (WB) analysis, and the experimental results are shown in FIG. 1: as can be seen from fig. 1, with the extension of ODN stimulation time, the level of TLR9 acetylation showed a gradual increase trend, and TSA was able to significantly enhance ODN-induced level of TLR9 acetylation, demonstrating that TLR9 has acetylation modification;
the exogenous experiment specifically comprises: in 293T cells, HA-TLR9 and HA-CBP plasmids are co-transfected, the cells are harvested after 48h, and immunoblot analysis is carried out after completion of co-immunoprecipitation, and the experimental results are shown in FIG. 2: as can be seen from fig. 2, acetyltransferase CBP mediates acetylation modification of TLR9, again demonstrating that TLR9 (i.e. protein a) has acetylation modification and that CBP (i.e. acetyltransferase B) is an acetyltransferase of TLR 9;
2) preparation of protein samples co-transfected with HA-TLR9 and HA-CBP and protein samples transfected with HA-TLR9 alone:
(1) the 5.2X10 cells were seeded on 20 6cm cell culture dishes 6293T cells, the next day when the cell density reaches 80%, using 24ul EZ Trans transfection reagent to independently transfect the cells of one cell culture dish with the plasmid 8ug of HA-TLR9, and simultaneously using 24ul EZ Trans transfection reagent to co-transfect the cells of the other 19 cell culture dishes with the plasmid HA-TLR9 and the plasmid HA-CBP of 4 ug;
(2) placing the transfected cell culture dish into an incubator at 37 deg.C and 5% CO2After the culture is carried out for 6 hours, the culture solution containing the EZ Trans-DNA compound is removed, and the culture solution is replaced by new culture solution for continuous culture48h；
(3) Collecting the cultured cells in a 1.5mL first centrifuge tube, centrifuging the cell precipitate at the rotation speed of 5000 revolutions at 4 ℃ for 10min, and adding 500ul of RIPA lysate into each first centrifuge tube;
(4) placing the first centrifuge tube on a vertical rotary shaking table, cracking at 4 ℃ for 1h, centrifuging at 4 ℃ for 20min at 20000 rpm, transferring the cell supernatant obtained by centrifugation into a 1.5mL second centrifuge tube, adding 4 μ L of HA antibody into the second centrifuge tube, and incubating on the vertical rotary shaking table overnight;
(5) the next day, 40ul Agarose beads (Protein G PLUS-Agarose) were added to the second centrifuge tube and incubated overnight at 4 ℃ on a vertical rotary shaker;
(6) centrifuging the antigen-antibody-agarose bead compound in the second centrifugal tube on the third day, washing with cold RIPA lysate for 3 times, and sucking dry liquid after the last washing;
(7) adding 20ul of loading buffer solution with the concentration two times of that of the obtained compound, boiling for 5min at 100 ℃, and then carrying out instantaneous centrifugation to obtain a single protein sample transfected with HA-A and 19 protein samples co-transfected with HA-TLR9 and HA-CBP, wherein the obtained protein samples can be directly used for WB analysis and can also be frozen in a refrigerator at-80 ℃ for later use;
3) from the above 19 protein samples co-transfected with HA-TLR9 and HA-CBP, 1 protein sample was randomly extracted and subjected to immunoblot analysis together with the protein sample transfected with HA-TLR9 alone, and the results of the detection analysis are shown in FIG. 3: as can be seen from FIG. 3, the protein samples co-transfected with HA-TLR9 and HA-CBP had undergone acetylation modification;
4) the remaining 18 protein samples co-transfected with HA-TLR9 and HA-CBP were run in five lanes on SDS-PAGE gel and incubated overnight in Coomassie Brilliant blue stain, and the gel was destained the next day on a shaker until the background of the gel was white and the destaining was stopped with the protein bands in the lanes, particularly the target band, blue, as shown in FIG. 4; and cutting a target strip according to the molecular weight of the A, putting the target strip into a third centrifuge tube, and marking to obtain the sample for identifying the specific protein acetylation modification.
The prepared sample for identifying the specific protein acetylation modification is sent to qualified biological companies for identification of TLR9 acetylation modification site mass spectrometry (LCMSMS), and the specific identification process comprises the following steps: and (3) performing enzymolysis, mass spectrometry data acquisition and data analysis on the test sample, and finally identifying 7 TLR9 acetylation modification sites, as shown in FIGS. 5-11.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (6)
1. A preparation method for identifying a specific protein acetylation modified sample is characterized by comprising the following steps:
1) selecting protein A with acetylation modification through an endogenous experiment, and determining acetyltransferase B of the protein A through an exogenous experiment;
2) preparation of protein samples of co-transfected plasmid a and plasmid B and protein samples of transfected plasmid a alone:
(1) respectively inoculating 293T cells in at least 20 cell culture dishes, and after the cell density reaches 78-82%, singly transfecting the plasmid A into cells in one cell culture dish by using a transfection reagent, and simultaneously co-transfecting the plasmid A and the plasmid B into the cells in the rest cell culture dishes by using the transfection reagent;
(2) placing the transfected cell culture dish into an incubator, culturing for 6 hours at constant temperature and constant carbon dioxide concentration, removing the culture solution containing the transfection reagent-DNA compound, and replacing a new culture solution to continuously culture for 48 hours;
(3) collecting the cultured cells in a first centrifuge tube, centrifuging cell precipitates, and adding RIPA lysate into the first centrifuge tube;
(4) placing the first centrifuge tube on a vertical rotary shaking table for cracking for 1h, centrifuging, transferring cell supernatant obtained by centrifuging into a second centrifuge tube, adding an antibody into the second centrifuge tube, and incubating on the vertical rotary shaking table overnight;
(5) adding agarose beads to the second centrifuge tube the next day, and then continuing to incubate overnight on a vertical rotary shaker;
(6) centrifuging the antigen-antibody-agarose bead compound in the second centrifugal tube on the third day, washing with cold RIPA lysate for 3 times, and sucking dry liquid after the last washing;
(7) adding a sample buffer solution with twice concentration into the obtained compound, boiling for 5-8min at 100 ℃, and then performing instantaneous centrifugation to obtain a protein sample of a single transfection plasmid A and at least 19 protein samples of co-transfection plasmids A and B;
3) randomly extracting 1 protein sample from the protein samples of the co-transfected plasmid A and the plasmid B, and completing immunoblotting analysis together with the protein sample of the single transfected plasmid A;
4) running SDS-PAGE gel on the protein samples of the rest cotransfection plasmids A and B, incubating in Coomassie brilliant blue staining solution overnight, decoloring the gel on a shaking table the next day until the background of the gel is white and the protein band, especially the target band, in the lane is blue, and stopping decoloring; and cutting a target strip according to the molecular weight of the A, putting the target strip into a third centrifuge tube, and marking to obtain the sample for identifying the specific protein acetylation modification.
2. The preparation method for identifying the specific protein acetylation modified sample according to claim 1, wherein: in the step 2) (1), the inoculation amount of the 293T cells is 5.2X106Per cell culture dish.
3. The preparation method for identifying the specific protein acetylation modified sample according to claim 1, wherein: in step 2) (1), the transfection reagent is an EZ Trans transfection reagent.
4. The preparation method for identifying the specific protein acetylation modified sample according to claim 1, wherein: in the step 2), the constant temperature is 37 ℃, and the concentration of the carbon dioxide is 5%.
5. The preparation method for identifying the specific protein acetylation modified sample according to claim 1, wherein: in step 2) (1), after the cell density reaches 80%, the plasmid A alone transfects the cells of one cell culture dish with the transfection reagent, and simultaneously, the plasmid A and the plasmid B co-transfect the cells of the rest of the cell culture dishes with the transfection reagent.
6. The preparation method for identifying the specific protein acetylation modified sample according to claim 1, wherein: in step 1), the exogenous experiment is as follows: in 293T cells, plasmid A and plasmid B were co-transfected, cells were harvested after 48h, co-immunoprecipitation was performed first, and then immunoblot analysis was performed.
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