CN109136166B - Method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis - Google Patents
Method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis Download PDFInfo
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Abstract
The invention belongs to the technical field of proteomics, and particularly discloses a method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis for the first time. The extraction method provided by the invention is simple to operate, high in extraction efficiency and low in cost, the purity of the obtained rice leaf plasma membrane can reach more than 93%, and the yield of plasma membrane phosphorylated protein enriched from the plasma membrane protein is 3.33-3.89%. The method is suitable for plant materials with difficult extraction of plasma membrane phosphorylated protein and much interference impurities; meanwhile, the obtained plasma membrane phosphorylated protein has fewer impurities and interferents, can meet the requirements of dimensional electrophoresis, and has clear background, uniform protein point distribution, less longitudinal trailing and horizontal cross striation phenomena and good repeatability; the method is suitable for preparing and analyzing the monocotyledon plasma membrane phosphorylated proteome.
Description
Technical Field
The invention relates to the technical field of proteomics, in particular to a method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis.
Background
Protein phosphorylation refers to phosphorylation of a protein by a protein kinase and subsequent phosphorylation by a phosphatase. Protein phosphorylation, one of the most important forms of post-translational modification of proteins, regulates almost all of the vital activities within a cell, and plays an extremely important role in regulating cell signaling processes in particular. The phosphoproteomics refers to the analysis method of proteomics, which is used for observing the modification state and the change of the phosphoprotein in cells on the whole so as to analyze the regulation and control effect of specific phosphorylation modification on the life process and the molecular mechanism of the phosphorylation modification; the content mainly comprises enrichment, detection, identification, quantification and the like of phosphorylated proteins. Among them, the 2-fractional gel electrophoresis (2-DE) technique is still the most mature and classical protein separation technique in the study of phosphoproteomics, and the research strategy is to purify and enrich the phosphoprotein first and then separate the phosphoprotein by electrophoresis and the like. The 2-DE technology has the advantages of high throughput, high resolution, good repeatability and the like, can clearly reflect the molecular weight and isoelectric point information of protein spots, and is one of the most commonly adopted methods in plant phosphorylation proteomics research.
Receptors and early reaction proteins which are related to the functions of recognizing various external stress signals and participating in signal transduction and the like exist on the plant cell plasma membrane, and the phosphorylation of the plasma membrane protein is proved to be widely involved in the reactions of signal recognition and the like to external stress (such as pathogenic bacteria and the like); therefore, the extraction and purification of rice cytoplasmic membrane phosphorylated protein are developed, which is beneficial to comprehensively understanding rice phosphorylated proteome, screening functional phosphorylated protein and the like. In the existing research on the plant plasma membrane phosphorylation proteome, the most adopted strategies are as follows: separating and purifying a plant plasma membrane, performing enzymolysis and phosphorylated peptide enrichment on plasma membrane protein, and performing protein phosphorylation modification analysis through chromatographic separation and mass spectrometry; its advantages are high solubility of peptide segment, high enriching specificity, and low cost.
At present, no reports related to rice plasma membrane phosphorylated proteome exist, and the main reasons are that the monocotyledon plasma membrane is difficult to purify, the abundance of plasma membrane phosphorylated protein is low, and the loss of protein is easily caused by the hydrophobicity of the plasma membrane protein. Therefore, it is necessary to develop a separation technology system for rice plasma membrane phosphorylated proteins suitable for dimensional electrophoresis analysis.
Disclosure of Invention
The invention aims to overcome the blank of the existing rice plasma membrane phosphorylated proteome separation and purification technology, and provides a method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis for the first time, which specifically comprises the steps of separation and purification of rice leaf plasma membrane, separation and purification of plasma membrane protein and Al (OH)3Incubation, washing of non-phosphorylated protein, elution of phosphorylated protein, acetone precipitation and the like, thereby obtaining the rice leaf plasma membrane phosphorylated protein with high purity.
Another objective of the invention is to provide an extraction buffer for rough rice leaf membranes.
Another objective of the invention is to provide a buffer solution for enriching the rice leaf plasma membrane phosphorylated protein.
In order to achieve the purpose, the invention is realized by the following scheme:
due to the characteristics of the phosphorylated protein itself, there are still some technical difficulties in the analysis of the phosphorylated protein: first, the abundance of most phosphorylated proteins in cells is low; secondly, the variability of phosphorylation, such that different forms of phosphorylation of a protein exist under different conditions; thirdly, the existing analysis method lacks dynamic analysis of phosphorylation sites, so that partial sites are difficult to identify; the hydrophobicity of the plasma membrane protein itself easily causes the loss of the protein; finally, the presence of phosphatase enzymes leads to dephosphorylation during sample preparation. Therefore, aiming at the problems, the invention optimizes and improves the existing phosphorylated protein extraction method and the buffer solution and the extracting solution used in the preparation process, so that the purity of the finally enriched plasma membrane phosphorylated protein is greatly improved, and the finally enriched plasma membrane phosphorylated protein contains fewer impurities and interferents and is suitable for the subsequent two-dimensional electrophoresis analysis.
Therefore, the invention claims an extraction buffer for rough membranes of rice leaves, which comprises the following components: BTP-MES buffer solution with a final concentration of 50mmol/L, sucrose with a final concentration of 250-500 mmol/L, EDTA with a final concentration of 0.5-10 mmol/L, 10-20% v/v glycerol, 0.1-1% w/v BSA with a final concentration of 1-10 mmol/LPMSF of 0.1-2 mmol/L of DTT, 0.6-5% w/v PVP k-30, NaF of 10-50 mmol/L of final concentration, beta-mercaptoethanol of 5-15 mmol/L of final concentration, NaMoO of 1-2 mmol/L of final concentration4·2H2O, NaVO with final concentration of 0.05-2 mmol/L3Sodium pyrophosphate with the final concentration of 2.5-10 mmol/L and 0.01-0.02% w/v Brij 56; the pH value of the rice leaf rough membrane extraction buffer solution is 7.6-7.8.
According to the invention, the components are preferably compounded to obtain the extraction buffer solution for the rough plasma membrane of the rice leaf, so that the degradation of plasma membrane protein can be effectively inhibited, the solubility of the plasma membrane protein is increased, and the loss of the protein is reduced. Wherein, EDTA and PMSF are protease inhibitors to prevent protein degradation; DTT, PVP k-30 and beta-mercaptoethanol are reducing agents, and disulfide bonds of protein molecules can be broken to increase the solubility of the protein; NaF, NaMoO4·2H2O、NaVO3The sodium pyrophosphate is a phosphatase inhibitor, can prevent dephosphorylation of target protein, is easy to remove, and does not influence subsequent two-dimensional electrophoresis analysis; poly (oxyethylene) cetyl ether, Brij56, is an ionic detergent that increases the solubility of plasma membrane proteins.
Preferably, the extraction buffer comprises the following components: BTP-MES buffer with a final concentration of 50mmol/L, sucrose with a final concentration of 250mmol/L, EDTA with a final concentration of 2mmol/L, 10% v/v glycerol, 0.5% w/v BSA, PMSF with a final concentration of 10mmol/L, DTT with a final concentration of 2mmol/L, 0.6% w/v PVP k-30, NaF with a final concentration of 40mmol/L, beta-mercaptoethanol with a final concentration of 15mmol/L, NaMoO with a final concentration of 1mmol/L4·2H2O, NaVO with final concentration of 1mmol/L3Sodium pyrophosphate at a final concentration of 10mmol/L, 0.02% w/v Brij56, pH 7.8.
The invention also claims a buffer solution for enriching the rice leaf plasma membrane phosphorylated protein, which comprises the following components: MES with the final concentration of 20-50 mmol/L, imidazole with the final concentration of 10-40 mmol/L, urea with the final concentration of 7-8 mol/L, potassium aspartate with the final concentration of 0.1-0.2 mol/L, sodium glutamate with the final concentration of 0.1-0.2 mol/L, 0.2-4% w/vCHAPS and the pH value of 6.0-6.3.
Wherein the miaowAzoles are phosphatase inhibitors; CHAPS is an ionic detergent and can improve the solubility of hydrophobic proteins in plasma membranes; the potassium aspartate and the sodium glutamate can enhance Al (OH)3The specific adsorption effect on the phosphorylated protein can improve the separation efficiency of the phosphorylated protein and the non-phosphorylated protein.
Preferably, the buffer comprises the following components: MES with a final concentration of 30mmol/L, imidazole with a final concentration of 30mmol/L, urea with a final concentration of 8mol/L, potassium aspartate with a final concentration of 0.1-0.15 mol/L, sodium glutamate with a final concentration of 0.1-0.15 mol/L, 0.25% w/v CHAPS and a pH value of 6.1.
The invention also claims a method for extracting the rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis, which comprises the following steps:
s1, extracting a rough membrane of a rice leaf: grinding the rice leaves into powder, adding the extraction buffer solution according to the mass-to-volume ratio of 4:13, and grinding until homogenate is obtained; filtering, centrifuging at 11500 Xg for 20min, centrifuging the supernatant at 87000 Xg for 40min, and suspending the precipitate in phase buffer to obtain crude membrane solution;
s2, purifying a rice leaf plasma membrane: adding the crude membrane solution obtained in the step S1 into a double aqueous phase system, uniformly mixing, centrifuging at 1000 Xg for 5min, taking the upper phase, and repeating for three times; centrifuging, collecting the upper phase solution, diluting with 10 times volume of phase buffer solution, centrifuging at 120000 Xg for 1h, and collecting precipitate; suspending the precipitate in plasma membrane preservation solution, centrifuging at 120000 Xg for 1h, and repeating twice; dissolving the precipitate with any one of the above buffer solutions to obtain purified plasma membrane protein;
s3, enrichment of rice leaf plasma membrane phosphorylated proteins: incubating the plasma membrane protein obtained in S2 at 20 deg.C for 2h, centrifuging at 15000 Xg for 15min, and collecting the supernatant; adding the supernatant to Al (OH)3And (4) incubating for 2 h; centrifuging at 6000 Xg for 10min, and collecting precipitate; adding any one of the above buffer solutions into the precipitate, mixing, centrifuging at 6000 Xg for 5min, removing supernatant, and repeating for 4 times; adding an elution buffer solution into the precipitate, uniformly mixing, and incubating at room temperature for 2 h; centrifuging at 10000 Xg for 5min, collecting supernatant, and enriching plasma membrane phosphorylated protein by DOC-TCA-acetone precipitation method.
The invention adopts a 6.3% w/w PEG 3350/Dextran T-500 two-phase distribution system to purify the rice leaf plasma membrane, and obtains a high-purity plasma membrane, wherein the purity is more than 93%; with Al (OH)3As a substrate, rice leaf plasma membrane phosphorylated protein is enriched, and the enrichment yield is 3.31-3.88%; the rice leaf plasma membrane phosphorylated protein is subjected to two-dimensional electrophoresis analysis, and through Pro-Q Diamond dyeing and image scanning, the 2-DE map of the rice leaf plasma membrane phosphorylated protein has good repeatability, clear background and uniform protein point distribution, and can meet the research requirements of subsequent analysis of phosphorylated proteomes; Pro-Q Diamond staining and silver nitrate staining are adopted to analyze the same 2-DE gel, and the result shows that the enriched rice leaf plasma membrane phosphorylated protein has high specificity.
In the process of extracting the rough rice leaf membranes, NaF and NaMoO are added4·2H2O、NaVO3Phosphatase inhibitors such as sodium pyrophosphate and imidazole can effectively inhibit the degradation of phosphorylated protein in the protein extraction process, and an ideal 2-DE map is obtained; in addition, because a large amount of hydrophobic protein exists in the plasma membrane and is difficult to dissolve, the ionic detergents Brij56, CHAPS and NP-40 are added in sequence in the processes of extracting and purifying the plasma membrane, so that the solubility of the plasma membrane protein is improved, and protein spots in an obtained 2-DE map are clear and uniformly distributed.
Preferably, the steps S1 and S2 are both performed at 4 ℃.
Preferably, the DOC-TCA-acetone precipitation method in S3 comprises the following specific steps: adding 2% w/v sodium deoxycholate into the supernatant, mixing uniformly, adding 100% w/v TCA, mixing uniformly, and standing on ice overnight; centrifuging at 18000 Xg for 15min, and removing supernatant; suspending and precipitating with 25% w/v TCA, centrifuging at 18000 Xg at 4 deg.C for 15min, and collecting precipitate; suspending the precipitate in 0.01mol/LTris-HCl solution containing 80% acetone at pH 7.5, and standing at-20 deg.C for more than 30 min; centrifuging at 4 deg.C for 10min, and removing supernatant; suspending the precipitate in 90% acetone, centrifuging at 4 deg.C for 10min at 20000 × g, and removing supernatant; and (5) placing the precipitate on ice for air drying to obtain the rice leaf plasma membrane phosphorylated protein. When the prepared rice leaf plasma membrane phosphorylated protein is used specifically, a solubilization buffer solution (7mol/L urea, 2mol/L thiourea, 2% v/v NP-40, 65mmol/L DTT) containing 1% w/v DTT is dissolved for standby.
Preferably, the phase buffers in S1 and S2 comprise the following final concentrations of the respective components: 5mmol/LK2HPO4-KH2PO4Buffer solution, 250mmol/L sucrose, 3mmol/L KCl, 1mmol/L DTT; the pH of the phase buffer was 7.8.
Preferably, the aqueous two-phase system in S2 comprises the following components: 6.3% w/w Dextran T-500/PEG 3350, sucrose to a final concentration of 250mmol/L, KCl to a final concentration of 3mmol/L, KH to a final concentration of 5mmol/L2PO4(ii) a The pH value of the aqueous two-phase system is 7.8.
Preferably, the plasma membrane preservation solution in S2 comprises the following components at the final concentrations: 5mmol/L BTP-MES buffer solution, 250mmol/L sucrose, 2mmol/L KCl, 1mmol/L DTT; the pH value of the plasma membrane preservation solution is 7.8.
Preferably, the elution buffer in S3 comprises the following final concentrations of the components: 0.3mol/L potassium pyrophosphate and 8mol/L urea; the pH of the elution buffer was 9.0.
Compared with the prior art, the invention has the following beneficial effects:
the extraction method provided by the invention is simple to operate, high in extraction efficiency and low in cost, the purity of the obtained rice leaf plasma membrane can reach more than 93%, and the yield of plasma membrane phosphorylated protein enriched from the plasma membrane protein is 3.33-3.89%. The method is suitable for plant materials with difficult extraction of plasma membrane phosphorylated protein and much interference impurities; meanwhile, the obtained plasma membrane phosphorylated protein has fewer impurities and interferents, can meet the requirements of dimensional electrophoresis, and has clear background, uniform protein point distribution, less longitudinal trailing and horizontal cross striation phenomena and good repeatability; the method is suitable for preparing and analyzing the monocotyledon plasma membrane phosphorylated proteome.
Drawings
FIG. 1 shows the binding of different specific inhibitors to rice leaf plasma membrane H in example 2+-effect of ATPase activity.
FIG. 2 is a silver nitrate staining 2-DE map of rice leaf plasma membrane phosphorylated protein in example 2.
FIG. 3 is the analysis of the goodness of fit of the rice leaf phosphorylated protein 2-DE profile in example 2 after staining by different methods; wherein A is the result of staining the gel with Pro-Q Diamond; b is the result of silver nitrate staining of the gel; c, marking the points in A as green by using analysis software; d is marking the points in the B picture as red using the analysis software; e is the result of superimposing C and D using the analysis software.
FIG. 4 is a Venn plot of the 2-DE profile protein spot goodness of agreement of Pro-Q Diamond staining with silver staining in example 2; wherein, yellow is the number of protein points of Pro-Q Diamond stained 2-DE map; green is the number of protein points of silver-stained 2-DE map.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 extraction method of phosphorylated protein from plasma membrane of rice leaf suitable for dimensional electrophoresis
1. Rice planting and leaf collection
The rice is indica rice line CO 39. The rice is sowed in a plastic pot containing rice field soil and is irradiated for 14 hours at 24-26 ℃ (about 250 mu mol.m)-2·s-1) Is grown in the growth chamber. And (4) performing sampling when the 4 th leaf is completely unfolded according to the management of a conventional method. Taking the 3 rd and 4 th leaves respectively, quickly weighing the leaves, quickly freezing the leaves by liquid nitrogen, and storing the leaves in a refrigerator at the temperature of 70 ℃ below zero for later use.
2. Extraction of rough rice leaf membranes
80g of rice leaves were weighed and ground to powder with liquid nitrogen. 260mL of extraction buffer (containing 50mmol/LBTP-MES buffer, 250mmol/L sucrose, 2mmol/L EDTA, 10% v/v glycerol, 0.5% w/v BSA, 10mmol/L PMSF, 2mmol/L DTT, 0.6% w/V-v PVP k-30, 40mmol/L NaF, 15mmol/L beta-mercaptoethanol, 1mmol/L NaMoO4·2H2O，1mmol/L NaVO310mmol/L sodium pyrophosphate, 0.02% w/v Brij56, ph7.8), ground to a homogenate. Filtering with a filter screen with aperture of 260 μm, centrifuging the filtrate at 11500 Xg for 20min, and collecting supernatant; the supernatant was centrifuged at 87000 Xg for 40min and the pellet was suspended in phase buffer (5 mmol/LK)2HPO4-KH2PO4Buffer solution, 250mmol/L sucrose, 3mmol/L LKCl, 1mmol/L DTT, pH7.8), and the obtained sample is the crude membrane solution. All the above operations were carried out at 4 ℃. Wherein EDTA and PMSF are protease inhibitors; DTT, PVP k-30 and beta-mercaptoethanol are reducing agents, and disulfide bonds of protein molecules can be broken to increase the solubility of the protein; NaF, NaMoO4·2H2O、NaVO3Sodium pyrophosphate is a phosphatase inhibitor; poly (oxyethylene) cetyl ether (Brij 56) is an ionic detergent that increases the solubility of plasma membrane proteins.
3. Purification of plasma membranes
Adding the above crude membrane solution into aqueous two-phase system (containing 6.3% w/w Dextran T-500/PEG 3350, 250mmol/L sucrose, 3mmol/L KCl, 5mmol/L KH)2PO4pH7.8), centrifuging at 1000 Xg for 5min, collecting the upper phase, and repeating for three times. Diluting the upper phase solution obtained by the last centrifugation with 10 times volume of phase buffer solution, centrifuging at 120000 Xg for 1h, and taking the precipitate; the pellet was suspended in plasma membrane stock (5mmol/L BTP-MES buffer, 250mmol/L sucrose, 2mmol/L KCl, 1mmol/L DTT, pH7.8), centrifuged at 120000 Xg for 1h, repeated twice; the precipitate was dissolved in incubation buffer (30mmol/LMES, 30mmol/L imidazole, 8mol/L urea, 0.1mol/L potassium aspartate, 0.1mol/L sodium glutamate, 0.25% w/v CHAPS) and the resulting sample was the purified plasma membrane. All the above operations were carried out at 4 ℃. Wherein imidazole is a phosphatase inhibitor; CHAPS is an ionic detergent and can improve the solubility of hydrophobic proteins in plasma membranes; the potassium aspartate and the sodium glutamate can enhance Al (OH)3Specific adsorption on phosphorylated proteins.
4. Enrichment of rice leaf plasma membrane phosphorylated protein
Will 8mg plasma membrane protein was incubated at 20 ℃ for 2h on a shaker, centrifuged at 15000 Xg for 15min and the supernatant was taken. The supernatant was added to 2g of Al (OH)3In the middle, incubating for 2h on a shaking table; centrifuging at 6000 Xg for 10min, and collecting precipitate. 15mL of washing buffer (30mmol/LMES, 30mmol/L imidazole, 8mol/L urea, 0.15mol/L potassium aspartate, 0.15mol/L sodium glutamate, 0.25% w/v CHAPS, pH 6.1) was added to the precipitate, vortexed, centrifuged at 6000 Xg for 5min, the supernatant removed, and repeated 4 times. Adding 20mL of elution buffer (0.3mol/L potassium pyrophosphate, 8mol/L urea, pH 9) into the precipitate, mixing uniformly, and incubating for 2h at room temperature in a shaking table; centrifuging at 10000 Xg for 5min, and collecting supernatant. Adding 2% w/v sodium deoxycholate, and mixing by vortex; then 100% w/v TCA was added, mixed well and left on ice overnight. Centrifuge at 18000 Xg for 15min, remove supernatant. Suspending and precipitating with 25% w/v TCA, and centrifuging at 18000 Xg for 15min at 4 deg.C; and taking the precipitate. Suspending the precipitate in 0.01mol/L Tris-HCl solution (pH 7.5, containing 80% acetone) and standing at-20 deg.C for more than 30 min; centrifuging at 4 deg.C for 10min, and removing supernatant; the pellet was resuspended in 1mL 90% acetone, centrifuged at 20000 Xg at 4 ℃ for 10min, and the supernatant removed. The precipitate was air dried on ice. The protein concentration was determined by dissolving it in a solubilization buffer containing 1% w/v DTT (7mol/L urea, 2mol/L thiourea, 2% v/v NP-40, 65mmol/L DTT) until use.
Example 2 two-dimensional electrophoresis analysis and specificity analysis of phosphorylated protein of rice leaf plasma membrane
1. Analysis of rice leaf plasma membrane purification effect
The rice leaf plasma membrane is purified by adopting a 6.3% w/w PEG 3350/Dextran T-500 two-phase distribution system, and the result shows that the yield of the plasma membrane protein obtained by purification is about 2.71-3.21% after 100mg of crude membrane protein is subjected to two-phase distribution for 3 times.
With different inner membrane marker enzymes (H)+ATPase) crude and purified plasma membranes were treated to evaluate the purity of the plasma membranes. The method comprises the following specific steps: mu.g of plasma membrane protein was added to 0.5mL of BTP-MES buffer (containing 30mmol/L of BTP-MES pH 6.5, 5mmol/L of MgSO4，50mmol/L KCl，0.02％(w/v)Brij 58，5mmol/L Na2-ATP), the reaction is started, incubated at 30 ℃ for 30min and 2.5mL of terminator [ containing 0.5 mmol-L PVP k30，86mmol/L(NH2OH)2·H2SO4，5.3mmol/L EDTA-Na20.2% v/v concentrated H2SO4，1.3％w/v(NH4)2MoO4]The reaction was terminated, and then 0.25mL of a developing solution (50mmol/L Na) was added rapidly2CO36.47mol/L NaOH) for 30min, and the absorption value at A720nm was measured; plasma membrane protein added with boiling for 30min was used as blank control. The activity inhibition rate is determined by adding 0.5mmol/L Na into the reaction system3VO4、50mmol/L KNO3、1mmol/L NaN3And 1mmol/L Na2MoO4Then, the absorbance at A720nm was measured, and H inhibited by each inhibitor was calculated+-the proportion of ATPase activity in the total activity. Wherein, Na3VO4Is plasma membrane H+-specific inhibitors of ATPase; KNO3Vacuolar membrane, endoplasmic reticulum membrane and Golgi apparatus membrane H+-specific inhibitors of ATPase; NaN3Is mitochondria and chloroplasts H+Inhibitors specific for ATPase, Na2MoO4Is lysosomal intimal H+Specific inhibitors of ATPase.
The results show (as shown in FIG. 1), that plasma membrane H+Sensitivity of ATPase to vanadate increased from 43.5% to 93.9%, H of type V (tonoplast, endoplasmic reticulum membrane, Golgi membrane, etc.)+-ATPase vs. KNO3The sensitivity of the strain is reduced from 66.8% to 8.2%, and the type F (mitochondria and chloroplasts) H+-ATPase vs. NaN3The sensitivity of the enzyme decreased from 71.9% to 6.4%, and the non-specific phosphatase (lysosome) was sensitive to Na2MoO4The sensitivity of (A) is reduced from 42.5% to 5.2%; the results show that the 6.3 percent PEG 3350/Dextran T-500 aqueous two-phase system can be used for effectively purifying the rough plasma membrane of the rice leaf, reducing other inner membrane impurities and obtaining a high-purity plasma membrane, wherein the purity is more than 93 percent.
2. Yield of rice leaf plasma membrane phosphorylated protein
Purified 8mg plasma membrane protein was mixed with 2g Al (OH)3Incubation, washing of non-phosphorylated proteins, elution of phosphorylated proteins and the likeAnd (3) enriching to obtain 265-310 mu g of plasma membrane phosphorylated protein, namely, the enrichment yield of the plasma membrane phosphorylated protein is 3.31-3.88%.
3. Two-dimensional electrophoresis analysis of rice leaf plasma membrane phosphorylated protein
In order to further determine whether the purified rice leaf plasma membrane phosphorylated protein is suitable for two-dimensional electrophoresis (2-DE), 2-DE and Pro-Q Diamond staining and image scanning were performed to obtain a 2-DE map of the rice leaf plasma membrane phosphorylated protein (shown in FIG. 2). The two-dimensional electrophoresis comprises the following steps: taking a plasma membrane phosphorylated protein sample (150 mu g) and hydrating in a solubilization buffer solution for 16h, and carrying out first phase gradient isoelectric focusing electrophoresis, wherein the focusing parameters are as follows: boosting the pressure at 300V for 0.5h at a low speed; boosting the pressure at a low speed of 500V for 0.5 h; boosting the pressure slowly for 0.5h at 1000V; rapidly boosting the pressure for 1.5h at 5000V; 8000V fast boost focus 60000 Vh. The concentration of the second SDS-PAGE was 12% and the electrophoresis parameter was 16 mA/gel. The same gel was stained sequentially by Pro-Q Diamond staining and silver staining. The gel was scanned using a Typhoon Trio multifunctional laser scanner.
PDQuest 8.0 software is used for analyzing the protein, and the result shows that 296 plasma membrane phosphorylated protein spots are in total in the 2-DE map and have good repeatability; meanwhile, the 2-DE map has clear background, protein spots are uniformly distributed, and the phenomena of longitudinal tailing and horizontal transverse striation are not obvious, so that the research requirement of subsequent analysis of the phosphorylated proteome can be met.
4. Specificity analysis of rice leaf plasma membrane phosphorylated protein
To examine the specificity of the method described in example 1 for the enrichment of plasma membrane phosphorylated proteins in rice leaf plasma membranes, Pro-Q Diamond staining and silver nitrate staining were performed on the same 2-DE gel, and overlay analysis was performed on the staining patterns of the same 2-DE gel by different methods using PDQuest 8.0 image analysis software (as shown in FIG. 3). The results show that: Pro-QDiamond stained plasma membrane phosphorylated protein spots were 296 (FIG. 3A) and total protein spots were 291 (FIG. 3B).
Protein spots in the Pro-Q Diamond staining pattern (FIG. 3A) were labeled green (FIG. 3C), and protein spots in the silver nitrate staining pattern (FIG. 3B) were labeled red (FIG. 3D); the two labeled maps were then superimposed (FIG. 3E) to analyze the goodness of fit of the protein spots in the two maps. The results show that: the overlay shows that most protein spots are in different shades of yellow, indicating that the protein is phosphorylated to a relatively high degree; part of protein spots are green, which shows that the protein of the protein spots is low in relative abundance and high in phosphorylation degree; the red color of only and individual protein spots indicates that the protein spot is a non-phosphorylated protein, or that the protein spot has a high relative abundance of protein and a low degree of phosphorylation (FIG. 4).
By combining the results, the rice leaf plasma membrane phosphorylated protein enriched by the extraction method of the invention has high specificity.
Comparative example 1
A method for extracting phosphorylated protein from plasma membrane of rice leaf, which is different from that in example 1 except that the extraction buffer solution adopted in the step 2 for extracting rough plasma membrane of rice leaf (lack of NaF and NaMoO)4·2H2O、NaVO3Sodium pyrophosphate), the rest steps are the same.
The formula of the buffer solution for extracting the rough plasma membrane of the rice leaf adopted in the comparative example is as follows: BTP-MES buffer at a final concentration of 50mmol/L, sucrose at a final concentration of 250mmol/L, EDTA at a final concentration of 2mmol/L, glycerol at a final concentration of 10% v/v, BSA at a final concentration of 0.5% w/v, PMSF at a final concentration of 10mmol/L, DTT at a final concentration of 2mmol/L, 0.6% w/v PVPk-30, beta-mercaptoethanol at a final concentration of 15mmol/L, 0.02% w/v Brij56, pH 7.8.
And (4) analyzing results: analysis of the purification effect of the rice leaf plasma membrane shows that the purity of the plasma membrane reaches 93.52%, the yield of the purified plasma membrane protein is about 2.94-3.02%, and the method has no obvious difference compared with the example 2. However, the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is only 2.18%, which is obviously lower than that of example 2. The result shows that the crude plasma membrane extraction buffer solution is lack of NaF and NaMoO4·2H2O、NaVO3And phosphatase inhibitors such as sodium pyrophosphate, plasma membrane phosphorylated proteins are degraded.
Comparative example 2
A method for extracting phosphorylated protein from a rice leaf plasma membrane comprises the same steps except that an extraction buffer used in the step 2 for extracting a rough rice leaf plasma membrane is different from that in the step 1 (NaF and sodium pyrophosphate are lacked).
The formula of the buffer solution for extracting the rough plasma membrane of the rice leaf adopted in the comparative example is as follows: BTP-MES buffer with a final concentration of 50mmol/L, sucrose with a final concentration of 250mmol/L, EDTA with a final concentration of 2mmol/L, 10% v/v glycerol, 0.5% w/vBSA, PMSF with a final concentration of 10mmol/L, DTT with a final concentration of 2mmol/L, 0.6% w/v PVP k-30, beta-mercaptoethanol with a final concentration of 15mmol/L, NaMoO with a final concentration of 1mmol/L4·2H2O, NaVO with final concentration of 1mmol/L30.02% w/v Brij56, pH 7.8.
And (4) analyzing results: analysis of the purification effect of the rice leaf plasma membrane shows that the purity of the plasma membrane reaches 93.47%, the yield of the purified plasma membrane protein is about 2.96-3.11%, and the method has no obvious difference compared with the example 2. However, the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is only 2.83%, which is obviously lower than that of example 2. NaF and sodium pyrophosphate are acid phosphatase inhibitors and have poor effect on alkaline phosphatase; the result shows that when the crude plasma membrane extraction buffer solution is lack of sodium fluoride and sodium pyrophosphate, the plasma membrane basic phosphorylated protein is easy to degrade.
Comparative example 3
A method for extracting phosphorylated protein from plasma membrane of rice leaf, which is different from that in example 1 except that the extraction buffer solution adopted in the step 2 for extracting rough plasma membrane of rice leaf (lack of NaF and NaMoO)4·2H2O、NaVO3Sodium pyrophosphate, Brij 56), the rest of the procedure was the same.
The formula of the buffer solution for extracting the rough plasma membrane of the rice leaf adopted in the comparative example is as follows: BTP-MES buffer with a final concentration of 50mmol/L, sucrose with a final concentration of 250mmol/L, EDTA with a final concentration of 2mmol/L, 10% v/v glycerol, 0.5% w/vBSA, PMSF with a final concentration of 10mmol/L, DTT with a final concentration of 2mmol/L, 0.6% w/v PVP k-30, beta-mercaptoethanol with a final concentration of 15mmol/L and a pH value of 7.8.
And (4) analyzing results: analysis of the purification effect of the rice leaf plasma membrane shows that the purity of the plasma membrane reaches 93.66%, and compared with example 2, the purity of the plasma membrane has no obvious difference. However, the yield of plasma membrane protein obtained by purification is about 2.18 to E2.35%, significantly lower than example 2. Meanwhile, the enrichment yield of the rice leaf plasma membrane phosphorylated protein is only 1.56%, which is obviously lower than that of the example 2. The results show that when the crude plasma membrane extraction buffer lacks the ionic detergent Brij56, the solubility of plasma membrane proteins is reduced, resulting in reduced plasma membrane protein yield; lack of NaF, NaMoO4·2H2O、NaVO3And phosphatase inhibitors such as sodium pyrophosphate, the phosphorylated protein is easily degraded, and the enrichment yield of plasma membrane phosphorylated protein is reduced.
Comparative example 4
A method for extracting phosphorylated protein from plasma membrane of rice leaf comprises the same steps except that step 3, the incubation buffer used in the purification of the plasma membrane is different from that used in example 1 (lacking imidazole, sodium glutamate and potassium aspartate).
The incubation buffer solution for enriching the rice leaf plasma membrane phosphorylated protein adopted by the comparative example comprises the following components: MES at a final concentration of 30mmol/L, urea at a final concentration of 8mol/L, 0.25% w/v CHAPS, pH 6.1.
And (4) analyzing results: the enrichment yield of the rice leaf plasma membrane phosphorylated protein is 3.90%, and compared with example 2, the enrichment yield has no obvious difference. However, SDS-PAGE results show that the prepared phosphorylated protein contains more impurities (non-phosphorylated protein), and the subsequent two-dimensional electrophoresis analysis results are influenced. Since imidazole, sodium glutamate and potassium aspartate can reduce Al (OH)3Non-specific adsorption of histidine-rich proteins and acidic amino acids, thus resulting in Al (OH) in the absence of the three components3The degree of specific adsorption to phosphorylated proteins is reduced, and the purity of the obtained phosphorylated proteins is also reduced.
Comparative example 5
A method for extracting phosphorylated protein from plasma membrane of rice leaf comprises the same steps except that the incubation buffer used in the purification of the plasma membrane in step 3 is different from that in example 1 (the concentrations of sodium glutamate and potassium aspartate are respectively 0.05 mol/L).
The incubation buffer solution for enriching the rice leaf plasma membrane phosphorylated protein adopted by the comparative example comprises the following components: MES with a final concentration of 30mmol/L, imidazole with a final concentration of 30mmol/L, urea with a final concentration of 8mol/L, potassium aspartate with a final concentration of 0.05mol/L, sodium glutamate with a final concentration of 0.05mol/L, 0.25% w/v CHAPS, pH 6.1.
And (4) analyzing results: the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is 2.89%, which is lower than that of the example 2. The results show that Al (OH) can be enhanced by potassium aspartate and sodium glutamate3Specific adsorption of phosphorylated proteins, therefore in the case of reduced potassium aspartate and sodium glutamate concentrations, Al (OH)3The specific adsorption degree of the phosphorylated protein is reduced, resulting in a reduction in the enrichment yield of the phosphorylated protein.
Comparative example 6
A method for extracting phosphorylated protein of rice leaf plasma membrane is the same as that in example 1 except that the washing buffer used in the step 4 for enriching the phosphorylated protein of rice leaf plasma membrane is different (the concentrations of sodium glutamate and potassium aspartate are respectively 0.20mol/L), and the other steps are the same.
The cleaning buffer solution for enriching the plasma membrane phosphorylated protein of the rice leaf adopted by the comparative example comprises the following components: MES with a final concentration of 30mmol/L, imidazole with a final concentration of 30mmol/L, urea with a final concentration of 8mol/L, potassium aspartate with a final concentration of 0.20mol/L, sodium glutamate with a final concentration of 0.20mol/L, 0.25% w/v CHAPS, pH 6.1.
And (4) analyzing results: the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is 3.01 percent, which is lower than that of the example 2. The results show that Al (OH) can be enhanced by potassium aspartate and sodium glutamate3Specific adsorption of phosphorylated proteins, and therefore increased concentrations of potassium aspartate and sodium glutamate, may result in binding to Al (OH)3The phosphorylated protein is difficult to elute, resulting in a decrease in the yield of the phosphorylated protein.
Comparative example 7
A method for extracting phosphorylated protein of a rice leaf plasma membrane comprises the same steps except that an elution buffer solution adopted in the enrichment of the phosphorylated protein of the rice leaf plasma membrane in the step 4 is different from that in the example 1 (0.15mol/L potassium pyrophosphate);
the elution buffer for enriching the plasma membrane phosphorylated protein of the rice leaf adopted in the comparative example comprises the following components: 0.15mol/L potassium pyrophosphate, 8mol/L urea, pH 9.
And (4) analyzing results: the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is 2.88 percent, which is lower than that of the example 2. Due to potassium pyrophosphate to Al (OH)3The substrate has strong affinity, and when the concentration of potassium pyrophosphate is reduced, the substrate may be bonded to Al (OH)3The phosphorylated protein is difficult to elute, resulting in a decrease in the yield of the phosphorylated protein.
It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the scope of the present invention, and those skilled in the art can make other variations or modifications based on the above description and ideas, and all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (8)
1. An extraction buffer solution for rough membranes of rice leaves is characterized by comprising the following components: BTP-MES buffer solution with a final concentration of 50mmol/L, sucrose with a final concentration of 250-500 mmol/L, EDTA with a final concentration of 0.5-10 mmol/L, glycerol with a concentration of 10-20% v/v, BSA with a concentration of 0.1-1% w/v, PMSF with a final concentration of 1-10 mmol/L, DTT with a final concentration of 0.1-2 mmol/L, PVP with a concentration of 0.6-5% w/v k-30, NaF with a final concentration of 10-50 mmol/L, beta-mercaptoethanol with a final concentration of 5-15 mmol/L, NaMoO with a final concentration of 1-2 mmol/L4·2H2O, NaVO with final concentration of 0.05-2 mmol/L3Sodium pyrophosphate with the final concentration of 2.5-10 mmol/L and 0.01-0.02% w/v Brij 56; the pH value of the rice leaf rough membrane extraction buffer solution is 7.6-7.8.
2. The extraction buffer according to claim 1, characterized in that it comprises the following components: BTP-MES buffer at a final concentration of 50mmol/L, sucrose at a final concentration of 250mmol/L, EDTA at a final concentration of 2mmol/L, 10% v/v glycerol0.5% w/v BSA, PMSF to a final concentration of 10mmol/L, DTT to a final concentration of 2mmol/L, 0.6% w/v PVPk-30, NaF to a final concentration of 40mmol/L, beta-mercaptoethanol to a final concentration of 15mmol/L, NaMoO to a final concentration of 1mmol/L4·2H2O, NaVO with final concentration of 1mmol/L3Sodium pyrophosphate at a final concentration of 10mmol/L, 0.02% w/v Brij56, pH 7.8.
3. A method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis is characterized by comprising the following steps:
s1, extracting a rough membrane of a rice leaf: grinding rice leaves into powder, adding the extraction buffer solution of any one of claims 1 or 2 according to a mass-to-volume ratio of 4:13, and grinding until homogenate is obtained; filtering, centrifuging at 11500 Xg for 20min, centrifuging the supernatant at 87000 Xg for 40min, and suspending the precipitate in phase buffer to obtain crude membrane solution;
s2, purifying a rice leaf plasma membrane: adding the crude membrane solution obtained in the step S1 into a double aqueous phase system, uniformly mixing, centrifuging at 1000 Xg for 5min, taking the upper phase, and repeating for three times; centrifuging, collecting the upper phase solution, diluting with 10 times volume of phase buffer solution, centrifuging at 120000 Xg for 1h, and collecting precipitate; suspending the precipitate in plasma membrane preservation solution, centrifuging at 120000 Xg for 1h, and repeating twice; dissolving the precipitate with rice leaf plasma membrane phosphorylated protein enrichment buffer solution to obtain purified plasma membrane protein;
s3, enrichment of rice leaf plasma membrane phosphorylated proteins: incubating the plasma membrane protein obtained in S2 at 20 deg.C for 2h, centrifuging at 15000 Xg for 15min, and collecting the supernatant; adding the supernatant to Al (OH)3And (4) incubating for 2 h; centrifuging at 6000 Xg for 10min, and collecting precipitate; adding rice leaf plasma membrane phosphorylated protein enrichment buffer solution into the precipitate, mixing, centrifuging at 6000 Xg for 5min, removing supernatant, and repeating for 4 times; adding an elution buffer solution into the precipitate, uniformly mixing, and incubating at room temperature for 2 h; centrifuging at 10000 Xg for 5min, collecting supernatant, and enriching plasma membrane phosphorylated protein by DOC-TCA-acetone precipitation method;
the rice leaf plasma membrane phosphorylated protein enrichment buffer comprises the following components: MES with the final concentration of 20-50 mmol/L, imidazole with the final concentration of 10-40 mmol/L, urea with the final concentration of 7-8 mol/L, potassium aspartate with the final concentration of 0.1-0.15 mol/L, sodium glutamate with the final concentration of 0.1-0.15 mol/L, 0.2-4% w/v CHAPS and the pH value of 6.0-6.3.
4. The extraction method according to claim 3, wherein the DOC-TCA-acetone precipitation method in S3 comprises the following steps: adding 2% w/v sodium deoxycholate into the supernatant, mixing uniformly, adding 100% w/v TCA, mixing uniformly, and standing on ice overnight; centrifuging at 18000 Xg for 15min, and removing supernatant; suspending and precipitating with 25% w/v TCA, centrifuging at 18000 Xg at 4 deg.C for 15min, and collecting precipitate; suspending the precipitate in 0.01mol/L Tris-HCl solution containing 80% acetone at pH 7.5, and standing at-20 deg.C for more than 30 min; centrifuging at 4 deg.C for 10min, and removing supernatant; suspending the precipitate in 90% acetone, centrifuging at 4 deg.C for 10min at 20000 × g, and removing supernatant; and (5) placing the precipitate on ice to be air-dried to obtain the rice leaf plasma membrane phosphorylated protein.
5. The extraction process according to claim 3, wherein the phase buffer in S1 and S2 comprises the following final concentrations of the respective components: 5mmol/L K2HPO4-KH2PO4Buffer solution, 250mmol/L sucrose, 3mmol/L KCl, 1mmol/L DTT; the pH of the phase buffer was 7.8.
6. The extraction method according to claim 3, wherein the aqueous two-phase system in S2 comprises the following components: 6.3% w/w Dextran T-500/PEG 3350, sucrose to a final concentration of 250mmol/L, KCl to a final concentration of 3mmol/L, KH to a final concentration of 5mmol/L2PO4(ii) a The pH value of the aqueous two-phase system is 7.8.
7. The extraction method according to claim 3, wherein the plasma membrane preservation solution in S2 contains the following components at final concentrations: 5mmol/L BTP-MES buffer, 250mmol/L sucrose, 2mmol/LKCl, 1mmol/L DTT; the pH value of the plasma membrane preservation solution is 7.8.
8. The extraction process according to claim 3, wherein the elution buffer in S3 comprises the following final concentrations of each component: 0.3mol/L potassium pyrophosphate and 8mol/L urea; the pH of the elution buffer was 9.0.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009059096A1 (en) * | 2007-10-31 | 2009-05-07 | E. I. Du Pont De Nemours And Company | Sequestration of formaldehyde to stabilize nitrilase specific activity when converting glycolonitrile to glycolic acid |
| CN101967181A (en) * | 2010-09-21 | 2011-02-09 | 南京农业大学 | Method for accumulating cotton phosphorylated protein |
| CN108181418A (en) * | 2017-12-18 | 2018-06-19 | 中国烟草总公司郑州烟草研究院 | A kind of P in Tobacco acidizing protein group determination method |
-
2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009059096A1 (en) * | 2007-10-31 | 2009-05-07 | E. I. Du Pont De Nemours And Company | Sequestration of formaldehyde to stabilize nitrilase specific activity when converting glycolonitrile to glycolic acid |
| CN101967181A (en) * | 2010-09-21 | 2011-02-09 | 南京农业大学 | Method for accumulating cotton phosphorylated protein |
| CN108181418A (en) * | 2017-12-18 | 2018-06-19 | 中国烟草总公司郑州烟草研究院 | A kind of P in Tobacco acidizing protein group determination method |
Non-Patent Citations (2)
| Title |
|---|
| Activation of Plant Plasma Membrane H+-ATPase by 14-3-3 Proteins Is Negatively Controlled by Two Phosphorylation Sites within the H+-ATPase C-terminal Region;Geoffrey Duby et al.;《JBC》;20090213;第284卷(第7期);第4214页右栏第4段 * |
| 水稻叶片质膜的纯化及质膜蛋白质双向电泳分析;何磊等;《华南农业大学学报》;20120115;第33卷(第1期);说明书第0043、0066-0076段、权利要求1 * |
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