CN113155577B - Preparation method for identifying specific protein acetylation modified sample - Google Patents
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Abstract
The invention belongs to the technical field of biology, 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 acetylase B; 2) Preparing protein samples of co-transfected plasmids A and B and protein samples of separately transfected plasmid A; 3) Selecting a protein sample of co-transfected plasmid A and plasmid B and separately transfecting the protein sample of plasmid A to complete immunoblotting analysis; 4) And (3) after the protein samples of the other co-transfected plasmids A and B are subjected to SDS-PAGE gel, incubating overnight in coomassie brilliant blue dye solution, decoloring, cutting off a target strip, and placing the target strip into a centrifuge tube to obtain the sample for identifying the specific protein acetylation modification. The protein sample prepared by the method can obviously enhance the success rate of identifying the specific protein acetylation modification site by mass spectrometry.
Description
[ field of technology ]
The invention relates to the field of biotechnology, in particular to a preparation method for identifying a specific protein acetylation modified sample.
[ background Art ]
Post-translational modification (Post-translational modification, PTM) of proteins refers to chemical modification of proteins after translation, and takes part in almost all normal vital activity processes of cells and plays a very important regulatory role. PTM is a means of protein function regulation and is critical to the structure and function of the protein. Studies have shown that 50% -90% of proteins in humans are post-translationally modified. More than 400 post-translational modifications have been identified, and common modifications include methylation, phosphorylation, ubiquitination, acetylation, glycosylation, SUMO, nitrosylation, oxidation, and the like. Therefore, PTM has become an extremely important field of international protein research. Among these, protein acetylation is a ubiquitous, reversible and highly regulated post-translational modification of proteins, mainly at the epsilon-NH 2 position of protein lysine residues. The acetylation modification is regulated by both acetyl transferase and deacetylase and is involved in almost all biological processes such as transcription, stress, metabolism and protein synthesis and degradation.
Detection of protein acetylation modification is different from phosphorylation, which can be studied by in situ phosphorylation of labels, sensitive phosphorylation-specific antibodies, etc. stable and effective means, and is relatively easy to detect. While the means and methods for detection of protein acetylation modification are very limited, these technical difficulties limit the research and development of protein acetylation.
The main methods for preparing and detecting protein lysine acetylation at present comprise the following steps: (1) Enriching the acetylated peptide fragment by using a lysine acetylation specific antibody, and detecting by using a liquid chromatography-mass spectrometry (HPLC/MS) method; (2) The identification of protein acetylation is completed by adopting a stable isotope labeled amino acid (stable isotope labeling with aminoacids in cell culture, SILAC) technology under the cell culture condition and a high-resolution and high-sensitivity electric field Orbitrap cyclotron resonance combination (LTQ Orbitrap) mass spectrometer. However, all of the above methods are based on proteomics level study of lysine acetylation modification, and cannot be used to detect the acetylation modification of a specific protein.
[ invention ]
In view of the foregoing, it is desirable to provide a method for identifying specific protein acetylation modified samples, wherein the protein samples prepared by the 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 above purpose, the technical scheme adopted by the invention is as follows:
a method of preparing a sample for identifying specific protein acetylation modifications, comprising the steps of:
1) Selecting protein A with acetylation modification through an endogenous experiment, and determining an acetylase B of the protein A through an exogenous experiment;
2) Protein samples of co-transfected plasmid a and plasmid B were prepared, and protein samples of plasmid a were transfected alone:
(1) Respectively inoculating 293T cells in at least 20 cell culture dishes, and when the cell density reaches 78-82%, independently transfecting the cells of one cell culture dish with the plasmid A, and simultaneously, co-transfecting the cells of the other cell culture dishes with the plasmid A and the plasmid B 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 complex, and replacing the new culture solution for continuous culturing for 48 hours;
(3) Collecting the cultured cells in a first centrifuge tube, centrifuging the cell sediment, and adding RIPA lysate into the first centrifuge tube;
(4) Placing the first centrifuge tube on a vertical rotary shaking table for cracking for 1 hour, centrifuging, transferring cell supernatant obtained by centrifuging into a second centrifuge tube, adding an antibody into the second centrifuge tube, and incubating on a vertical rotary shaking table overnight;
(5) Adding agarose beads into the second centrifuge tube the next day, and then continuously incubating on a vertical rotary table for overnight;
(6) Centrifuging the antigen-antibody-agarose bead complex in the second centrifuge tube on the third day, washing 3 times with cold RIPA lysate, and sucking the liquid after the last washing;
(7) Adding a double-concentration loading buffer solution into the obtained compound, boiling at 100 ℃ for 5-8min, and performing instantaneous centrifugation to obtain a protein sample of the single transfected plasmid A and protein samples of at least 19 co-transfected plasmids A and B;
3) Randomly extracting 1 protein sample from the protein samples of the cotransfected plasmid A and the plasmid B, and performing immunoblotting analysis together with the protein sample of the cotransfected plasmid A alone;
4) After running the remaining protein samples of co-transfected plasmid a and plasmid B on SDS-PAGE gel, incubating overnight in coomassie blue dye solution, the next day the gel was decolorized on a shaker until the background of the gel was white and the protein bands in the lanes, particularly the target bands, were blue and the decolorization was terminated; and cutting off 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 step (1) of step 2), the 293T cells were seeded at an amount of 5.2X10 6 The/each cell culture dish.
Further, in step (1) of step 2), the transfection reagent is an EZ Trans transfection reagent.
Further, in step (2) of step 2), the constant temperature is 37 ℃, and the concentration of carbon dioxide is 5%.
Further, in step (1) of step 2), after the cell density reaches 80%, plasmid A alone transfects cells of one cell culture dish with the transfection reagent, while plasmid A and plasmid B are co-transfected with the transfection reagent into cells of the remaining cell culture dish.
Further, in step 1), the exogenous experiment is: plasmid a and plasmid B were co-transfected in 293T cells, cells were harvested after 48h, first subjected to co-immunoprecipitation and then subjected to immunoblot analysis.
The invention has the following beneficial effects:
firstly, determining that the selected protein has acetylation modification through an endogenous experiment, further determining that the selected protein has acetylation modification through an exogenous experiment, determining that the accuracy reaches 100% in a double manner, and determining that the protein has acetylation transferase through the exogenous experiment; secondly, the protein samples which are subjected to and not subjected to acetylation modification are effectively enriched through an IP technology; thirdly, before mass spectrum identification is carried out on samples, the invention ensures that all samples to be detected are subjected to acetylation modification through immunoblotting analysis, and invalid samples are avoided; fourth, protein samples identified by mass spectrometry are tailored such that they have sufficient abundance to more readily identify sites of acetylation modification. Through the organic combination of the steps, the protein sample prepared by the preparation method can obviously enhance the success rate of identifying the specific protein acetylation modification site by mass spectrometry.
[ description of the drawings ]
FIG. 1 is a graph showing the results of the present invention ODN inducing an acetylation modification of endogenous TLR9 (i.e., graph showing the results of endogenous experiments).
Fig. 2 is a graph of the detection results of exogenous acetylation modification of TLR9 of the invention (i.e., exogenous experimental results).
FIG. 3 is a graph showing the results of an acetylation modification assay performed on randomly sampled protein samples using immunoblot analysis techniques.
FIG. 4 is a Coomassie brilliant blue staining chart of protein samples after running gel co-transfected with HA-A and HA-B.
Fig. 5-11 are secondary mass spectra of TLR9 acetylation sites.
The invention will be further described in the following detailed description in conjunction with the above-described figures.
[ detailed description ] of the invention
In a preferred embodiment of the invention, the identification of the site of the acetylation modification of TLR9 is exemplified.
A method of preparing a sample for identifying specific protein acetylation modifications, comprising the steps of:
1) Determining that the TLR9 has acetylation modification through an endogenous experiment, and determining the acetylase CBP of the TLR9 through an exogenous experiment;
wherein, endogenous experiments specifically are: mice macrophages raw264.7 were stimulated with the same concentration of TLR9 receptor activator ODN (0.5 μm) at different time points (0 min, 15min, 30 min), and the above cells were stimulated with the addition of a deacetylase inhibitor (Trichostatin a, TSA) based on ODN stimulation for 30min, and then the cells were collected for Immunoprecipitation (IP) and immunoblot (WB) analyses, the experimental results are shown in fig. 1: as can be seen from fig. 1, along with the extension of the ODN stimulation time, the TLR9 acetylation level showed a gradual increase trend, and TSA can significantly enhance the ODN-induced TLR9 acetylation level, demonstrating that TLR9 has an acetylation modification;
the exogenous experiment is specifically as follows: in 293T cells, HA-TLR9 and HA-CBP plasmids were co-transfected, cells were harvested after 48h, immunoblot analysis was performed after co-immunoprecipitation was completed, and experimental results are shown in FIG. 2: from fig. 2, it can be seen that the acetylase CBP mediates the acetylating modification of TLR9, again demonstrating that TLR9 (i.e. protein a) has an acetylating modification and that CBP (i.e. acetylase B) is an acetylase of TLR 9;
2) Protein samples co-transfected with HA-TLR9 and HA-CBP were prepared, protein samples transfected with HA-TLR9 alone:
(1) Inoculating 5.2X10 to 20 6cm cell culture dishes 6 The next day when the cell density reached 80%, cells of one of the cell culture dishes were transfected with plasmid 8ug of HA-TLR9 alone with 24ul of EZ Trans transfection reagent, while cells of the remaining 19 cell culture dishes were co-transfected with 4ug of plasmid HA-TLR9 and 4ug of plasmid HA-CBP with 24ul of EZ Trans transfection reagent;
(2) Placing the transfected cell culture dish into an incubator, and adding CO with concentration of 5% at 37 DEG C 2 After culturing for 6 hours, removing the culture solution containing the EZ Trans-DNA complex, and replacing the new culture solution for culturing for 48 hours;
(3) Collecting the cultured cells in a 1.5mL first centrifuge tube, centrifuging the cell sediment at a rotation speed of 5000 revolutions at 4 ℃ for 10min, and adding 500ul of RIPA lysate into each first centrifuge tube;
(4) Placing the first centrifugal tube on a vertical rotary shaking table, cracking for 1h at 4 ℃, centrifuging for 20min at 20000 revolutions at 4 ℃, transferring the cell supernatant obtained by centrifugation into a 1.5mL second centrifugal tube, adding 4 mu L of HA antibody into the second centrifugal tube, and incubating on the vertical rotary shaking table overnight;
(5) After the next day 40ul of Agarose beads (Protein G PLUS-Agarose) was added to the second centrifuge tube, incubated overnight on a vertically rotating shaker at 4 ℃;
(6) Centrifuging the antigen-antibody-agarose bead complex in the second centrifuge tube on the third day, washing 3 times with cold RIPA lysate, and sucking the liquid after the last washing;
(7) Adding 20ul of twice-concentration loading buffer solution into the obtained compound, boiling for 5min at 100 ℃, and performing instantaneous centrifugation to obtain a protein sample of separately transfected HA-A and 19 protein samples of co-transfected HA-TLR9 and HA-CBP, wherein the obtained protein samples can be directly used for WB analysis or frozen in a refrigerator at-80 ℃ for later use;
3) 1 protein sample was randomly extracted from the above 19 protein samples co-transfected with HA-TLR9 and HA-CBP, and immunoblot analysis was performed together with the protein sample transfected with HA-TLR9 alone, and the detection analysis results are shown in FIG. 3: as can be seen from fig. 3, protein samples co-transfected with HA-TLR9 and HA-CBP have undergone acetylation modification;
4) After the remaining 18 protein samples co-transfected with HA-TLR9 and HA-CBP were placed on five lanes on an SDS-PAGE gel to start running, the gel was decolorized on a shaker the next day after incubation overnight in coomassie blue dye solution until the background of the gel was white and the protein bands in the lanes, in particular the band of interest, were blue and the decolorization terminated, the results are shown in fig. 4; and cutting off 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 company for TLR9 acetylation modification site mass spectrometry (LCMS) identification, and the specific identification process comprises the following steps: the sample enzymolysis, mass spectrometry data acquisition and data analysis finally identify 7 TLR9 acetylation modification sites, as shown in figures 5-11.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.
Claims (6)
1. A method for preparing a sample for identifying specific protein acetylation modifications, comprising the steps of:
1) Selecting protein A with acetylation modification through an endogenous experiment, and determining an acetylase B of the protein A through an exogenous experiment;
2) Protein samples of co-transfected plasmid a and plasmid B were prepared, and protein samples of plasmid a were transfected alone:
(1) Respectively inoculating 293T cells in at least 20 cell culture dishes, and when the cell density reaches 78-82%, independently transfecting the cells of one cell culture dish with the plasmid A, and simultaneously, co-transfecting the cells of the other cell culture dishes with the plasmid A and the plasmid B 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 complex, and replacing the new culture solution for continuous culturing for 48 hours;
(3) Collecting the cultured cells in a first centrifuge tube, centrifuging the cell sediment, and adding RIPA lysate into the first centrifuge tube;
(4) Placing the first centrifuge tube on a vertical rotary shaking table for cracking for 1 hour, centrifuging, transferring cell supernatant obtained by centrifuging into a second centrifuge tube, adding an antibody into the second centrifuge tube, and incubating on a vertical rotary shaking table overnight;
(5) Adding agarose beads into the second centrifuge tube the next day, and then continuously incubating on a vertical rotary table for overnight;
(6) Centrifuging the antigen-antibody-agarose bead complex in the second centrifuge tube on the third day, washing 3 times with cold RIPA lysate, and sucking the liquid after the last washing;
(7) Adding a double-concentration loading buffer solution into the obtained compound, boiling at 100 ℃ for 5-8min, and performing instantaneous centrifugation to obtain a protein sample of the single transfected plasmid A and protein samples of at least 19 co-transfected plasmids A and B;
3) Randomly extracting 1 protein sample from the protein samples of the cotransfected plasmid A and the plasmid B, and performing immunoblotting analysis together with the protein sample of the cotransfected plasmid A alone;
4) After running the remaining protein samples of co-transfected plasmid a and plasmid B on SDS-PAGE gel, incubating overnight in coomassie blue dye solution, decolorizing the gel on a shaker the next day until the background of the gel is white and the protein band in the lane is blue and stopping decolorizing; and cutting off 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 method of claim 1, wherein the method comprises the step of: in step (1) of step 2), the 293T cells were seeded at a rate of 5.2X10 6 The/each cell culture dish.
3. The method of claim 1, wherein the method comprises the step of: in step (1) of 2), the transfection reagent is an EZ Trans transfection reagent.
4. The method of claim 1, wherein the method comprises the step of: in step (2) of step 2), the constant temperature is 37 ℃, and the concentration of carbon dioxide is 5%.
5. The method of claim 1, wherein the method comprises the step of: in step (1) of step 2), after the cell density reaches 80%, plasmid A alone transfects cells of one cell culture dish with the transfection reagent, while plasmid A and plasmid B are co-transfected with the transfection reagent into cells of the remaining cell culture dish.
6. The method of claim 1, wherein the method comprises the step of: in step 1), the exogenous experiment is: plasmid a and plasmid B were co-transfected in 293T cells, cells were harvested after 48h, first subjected to co-immunoprecipitation and then subjected to immunoblot analysis.
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| WO2003102196A1 (en) * | 2002-05-30 | 2003-12-11 | Centre National De La Recherche Scientifique (Cnrs) | Vectors for expression of biotinylated proteins in mammalian cells, and their use for identification of protein-nucleic acid interactions in vivo |
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