Total Protein Extraction with TCA-Acetone

TCA-Acetone Extraction 1 1 Total Protein Extraction with TCA-Acetone Valérie Méchin, Catherine Damerval, and Michel Zivy Summary We describe a procedure allowing extraction of total proteins that performs efficiently with a large variety of plant tissues, based on simultaneous precipitation and denaturation with TCA and 2ME in cold acetone. We also describe protein solubilization prior to IEF, either in classical rod gels or in IPGs, using two different solutions. The procedure is easy to carry out. The major caveats are (1) keep samples at low temperature during extraction, and then (2) manage protein samples at about 22 to 25 C to avoid urea precipitation. Key Words: protein extraction; protein solubilization; TCA; acetone; total proteins; chaotropes; detergents; reducing agents; plant proteomic. 1. Introduction In the context of proteomic studies, comparison of 2D gels requires wellresolved proteins; streaking and smearing must be avoided, as well as artifacts caused by proteolysis. Protein patterns must be reproducible from gel to gel. Sample preparation is thus a crucial step prior to electrophoresis. The main difficulties with plant tissues are low cellular protein content, the presence of proteases and interfering compounds such as phenolics, pigments, lipids, nucleic acids, and others (1). After extracting as many different proteins as possible, one has to solubilize them in a solution compatible with isoelectric focusing (IEF). After working with a large variety of plant tissues, we developed a method in which proteins are denatured and precipitated in a mixture of 2-mercaptoethanol (2ME) and trichloracetic acid (TCA) in cold acetone. It was derived from the method of Wu and Wang (2), who showed that TCA precipitation efficiently inhibits protease activity in plant tissues. Proteins were then solubilized in the urea-k 2 CO 3 -sodium dodecyl sulfate (SDS) (UKS) solution (3). This procedure gives highly reproducible gels with a good spot resolution in large From: Methods in Molecular Biology, vol. 335: Plant Proteomics: Methods and Protocols Edited by: H. Thiellement, M. Zivy, C. Damerval, and V. Méchin Humana Press Inc., Totowa, NJ 1

2 Méchin, Dameval, and Zivy ph and Mr ranges, and it has become very popular under the name of the TCA/ acetone method (4,5). The UKS solubilization solution was developed for the first dimension in IEF rod gels: the ph gradient was generated by carrier ampholytes upon voltage application. The replacement of these classical gels by immobilized ph gradient gels (IPGs) significantly improved the reproducibility of the first-dimension separation. At the end of the 1990s, the development of commercially available large-size IPGs led to their wide adoption as IEF media. However, running protein samples on IPGs prompted modifications in solubilization procedures, because of high salt content and the presence of ionic detergent (SDS) in UKS that are not compatible with the high voltages required to perform separation in such gels (6). We describe the different steps of our procedure, from protein precipitation and denaturation to resolubilization in a solution suitable for subsequent IEF in IPGs. 2. Materials 1. Precipitation solution: 10% TCA (w/v), 0.07% 2ME (v/v) in cold acetone. This solution must be freshly prepared and stored at 20 C until use (see Note 1). Caution: All three components are toxic, and the solution must be prepared under a hood on. 2. Rinsing solution: 0.07% 2ME (v/v) in cold acetone. This solution can be stored at 20 C for about 1 mo. 3. R2D2 solubilization solution: 5 M urea, 2 M thiourea, 2% 3[3-cholaminopropyl diethylammonio]-1-propane sulfonate (CHAPS; w/v), 2% N-decyl-N,N-dimethyl- 3-ammonio-1-propane sulfate (SB3-10) (w/v), 20 mm dithiothreitol (DTT), 5mM phosphine, 0.5% Pharmalyte 4-6.5 (v/v), 0.25% Pharmalyte 3-10 (v/v) in doubledistilled (dd)h 2 O. The solution is slightly heated (below 30 ; see Note 2) to aid urea solubilization. It is aliquoted and stored at 80 C for months (see Note 3). 4. UKS solubilization solution: 9.5 M urea, 5 mm K 2 CO 3, 1.25% SDS, 5% DTT, 6% Triton X-100, 2% ampholines 3.5 to 9.5 in ddh 2 O. K 2 CO 3 is prepared as a 2.8 % (w/v) stock solution and SDS as a 10% (w/v) filtered stock solution, Triton X-100 is provided as a 20% solution. Then 3 ml ddh 2 O are added to the other components until the urea is solubilized (by heating below 30 C; see Note 2). Solutions of 40 ml are usually prepared and then aliquoted and stored at 80 C for months. 5. Determination of protein concentration: The protein content of the samples (either in UKS or R2D2) is evaluated using the 2-D Quant Kit from Amersham Biosciences. The method involves a precipitation of proteins from the sample, followed by specific binding of copper ions to proteins as described by the manufacturers instructions (ref. 80-6483-56). The procedure is compatible with common sample preparation reagents. 6. IPG strip rehydration solution (for UKS only): a. Solution A: 7 M urea, 2 M thiourea, 1.4% CHAPS (w/v), 16 mm DTT, 5 mm phosphine, 0.3% Pharmalyte 3-10 (v/v) in ddh 2 O.

TCA-Acetone Extraction 3 b. Solution B: 7 M urea, 2 M thiourea, 0.5% CHAPS (w/v), 10 mm DTT, 5 mm phosphine, in ddh 2 O. 3. Methods 3.1. Protein Precipitation and Denaturation 1. Grind the plant tissues in a mortar and pestle in liquid nitrogen to obtain a fine powder (see Note 4). 2. Transfer about 200 μl of powder to a weighed 2-mL Eppendorf tube. Cover with 1.8 ml of the cold TCA-2ME-acetone solution (see Note 5), mix, and then store at 20 C for 1 h. TCA and acetone denature and precipitate the proteins. The solution inactivates the phenoloxidases and oxidases, preventing phenol oxidation into quinones, which would result in protein binding into insoluble complexes. It has also been shown to inactivate proteases (3), as well as phenol extraction (7,8). Acetone alone allows solubilization of the pigments, lipids, and terpenoids possibly present in the tissue. 2ME prevents the formation of disulfide bonds during precipitation. 3. Centrifuge for 10 min at 10,000g (in a refrigerated centrifuge, below 4 C). 3.2. Rinsing with 2ME-Acetone Solution 1. Discard the supernatant and resuspend the pellet in 1.8 ml of cold rinsing solution (see Note 5). Store at 20 C for 1 h. This step eliminates the acidity caused by TCA that would impede protein recovery. 2. Centrifuge for 15 min at 10,000g (in a refrigerated centrifuge, below 4 C). 3. Discard the supernatant. This step is repeated twice (see Note 6). 4. Dry the pellet under vacuum for about 1 h to eliminate the acetone fully (alternatively, dry for 20 30 min in a SpeedVac without heating). 5. Weigh the pellet (see Note 7). 3.3. Protein Solubilization 1. The amount of UKS or R2D2 buffers for protein solubilization depends on the plant tissue; for instance, we use 60 μl/mg dry powder for leaf tissue (maize, rape) and 50 μl/mg dry powder for maize kernels.- 2. Resolubilization is achieved by vortexing for 1 min. At this stage the sample still contains cellular debris. 3. Centrifuge for 15 min at 10,000g (25 C), and collect the supernatant in a 1.5-mL Eppendorf tube. 4. Centrifuge again (15 min, 25 C), and transfer the supernatant to a new Eppendorf tube. Samples containing solubilized proteins can then be stored at 50 C or 80 C for months. Solutions used to resuspend and solubilize proteins generally contain chaotropes, detergents, and reducing agents (9). Chaotropes unfold proteins by breaking noncovalent bonds. The most commonly used chaotropes are urea and thiourea. They are often used in combination, which improves protein solubili-

4 Méchin, Dameval, and Zivy zation. Detergents are needed to improve protein solubilization in the presence of chaotropic agents. CHAPS, a zwitterionic detergent, has solubilizing properties similar to those of Triton X-100 (a nonionic detergent) but has a more powerful effect in preventing protein-protein interaction. SB3-10 is even more efficient than CHAPS in solubilizing protein, but is poorly soluble at high concentration in urea. Thiol reducing agents (DTT is the most commonly used) and phosphines prevent disulfide bond formation. Phosphine in the form of tris carboxyethyl phosphine (TCEP)-HCl (which is nonvolatile, stable, and soluble in aqueous solutions, contrary to phosphine in the form of TBP) is known to be more selective and efficient than DTT, since it keeps its reducing power at acidic ph and at ph above 7.5. The R2D2 solution, which was specifically designed for solubilization of proteins prior to IEF in IPGs (6), associates urea with thiourea (see Note 2), CHAPS with SB3-10, and DTT with phosphine. The UKS solution was developed earlier, before the finding that the addition of thiourea and zwitterions could increase protein solubilization. Thus, it contains high-molarity urea (close to saturation; see Note 2) instead of a mixture of urea and thiourea and Triton X-100 instead of CHAPS and SB3-10. The specificity of UKS relies mainly on the presence of the ionic detergent SDS. SDS greatly helps the solubilization of proteins and, probably because of its ability to denaturate proteins, it limits the activity of proteases that may occur even in the presence of urea at saturation (10). The presence of SDS in the sample is in principle incompatible with IEF because it is a ionic detergent, but it is very well tolerated at this concentration, probably because of the simultaneous presence of Triton X-100, with which it can form micelles that migrate to the cathodic end of the IEF gel. UKS also contains K 2 CO 3, which alkalizes the buffer and therefore limits protein-protein and protein nucleic acid interactions, as well as protease activity. R2D2 is easier to use than UKS, because it is used for protein solubilization and IPG strip rehydration; a different rehydration solution must be prepared for UKS-solubilized samples, to compensate for the excess of SDS and other compounds. As few proteins seem to be more soluble in one or the other of these two solutions, it is recommended to stick with one. A priori UKS should be preferred only in samples in which high levels of protease activity are suspected. In both UKS and R2D2 solutions, adding ampholytes improves the resolution in the IEF dimension. 3.4. Preparation of Samples for IEF 1. Protein samples must be centrifuged once again before use and the pellet discarded.

TCA-Acetone Extraction 5 5 Fig. 1. 2-D gels obtained using R2D2 to solubilize proteins extracted following the TCA/acetone procedure. (A,B) Maize leaf and 14 DAP maize endosperm protein gels, respectively. (C,D) Rape stem and rape root protein gels, respectively. First 50 μg (A,B) or 250 μg of proteins (C,D) are loaded onto IPG (ph 4 7 linear) using the rehydration loading method. Passive rehydration is allowed for 1 h. Then active rehydration is performed at 22 C for 12 h at 50 V. IEF is achieved by 0.5 h at 200 V, 0.5 h at 500 V, 1 h at 1000

6 Méchin, Dameval, and Zivy 6 Fig. 1. (continued) V, and 10,000 V for the duration to reach 84,000 Vh. After IEF, IPG strips are equilibrated and then sealed at the top of the 1-mm-thick second-dimensional gel with the help of 1% low melting agarose. We use continuous 11% T, 2.67% C homemade gels with piperazine diacrylamide (PDA) as the crosslinking agent. After SDS-polyacrylamide gel electrophoresis (PAGE), proteins are visualized by silver staining (A,B) or colloidal Coomassie Blue G250 staining (C,D).

TCA-Acetone Extraction 7 2. About 50 μg of total proteins are loaded per IEF gel (24 cm long, either classical rod gels or IPGs) for analytical gels revealed with silver nitrate (Fig. 1A and B), or 150 to 500 μg for gels revealed with colloidal Coomassie blue (Fig. 1C and D). 3. Samples solubilized in R2D2 are ready to use in IEF with IPGs. One has just to complement the necessary volume of solubilized sample to reach the desired protein amount with the R2D2 solubilization buffer, up to 450 μl for active rehydration (6). Samples solubilized in UKS are ready to use in IEF with classical rod gels and ampholytes (3). For IEF in IPGs, they must be complemented up to 450 μl with another solution (A or B; see Note 8), which brings in thiourea, CHAPS, and phosphine, thus improving resolution by lowering the proportion of salt and SDS in the sample. 4. Notes 1. The precipitation and rinsing solutions must be cold when used. Always keep an acetone bottle at 4 C to prepare these solutions. 2. Both UKS and R2D2 solutions contain high molarity of urea and must not be heated above 30 C (which may be tempting to solubilize urea), because isocyanate ions are produced that would result in protein carbamylation. 3. SB3-10 is poorly soluble in urea (this is why the urea concentration has been limited to 5 M), and it is necessary to be patient when melting a frozen R2D2 aliquot. An aliquot can be efficiently used when the solution is perfectly limpid (i.e., when all R2D2 constituants are solubilized). 4. A fine powder must be obtained for efficient protein extraction. This may require precrushing of hard material (e.g., mature maize grains). It is also possible to use an automatic cryogenic crusher with a metallic ball (6 mm diameter). 5. Up to pellet drying, it is important to work at a low temperature (below 4 C) to limit protease action. 6. Several rinsing steps can profitably be applied with highly pigmented samples, so that a white pellet is eventually recovered. It is possible to extend rinsing overnight. 7. It is possible to store the dry pellet powder at 80 C before protein solubilization. However, in this case, it is better to redry the powder before protein resolubilization. 8. According to the volume of sample necessary to reach the desired protein amount, solution A or B is used. When 20 to 90 μl are sufficient, solution A is used to complement the solution of UKS-solubilized proteins. When the required volume is higher, solution B is used for complementation. We thus obtain suitable ionic conditions for IEF in IPGs. References 1. Damerval, C., Zivy, M., Granier, F., and de Vienne, D. (1988) Two-dimensional electrophoresis in plant biology, in Advances in electrophoresis (Chrambach, A., Dunn, M., and Radola, B., eds.), VCH, Weinheim, New York, pp. 265 340.

8 Méchin, Dameval, and Zivy 2. Wu, F. and Wang, M. (1984) Extraction of proteins for sodium dodecyl sulfate polyacrylamide gel electrophoresis from protease-rich plant tissues. Anal. Biochem. 139, 100 103. 3. Damerval, C., de Vienne, D., Zivy, M., and Thiellement, H. (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat seedling proteins. Electrophoresis 7, 52 54. 4. Saravanan, R. S. and Rose, J. K. (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4, 2522 2532. 5. Carpentier, S. C., Witters, E., Laukens, K., Deckers, P., Swennen, R., and Panis, B. (2005) Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5, 2497 2507. 6. Méchin, V., Consoli, L., Le Guilloux, M., and Damerval, C. (2003) An efficient solubilization buffer for plant proteins focused in immobilized ph gradients. Proteomics 3, 1299 1302. 7. Hurkman, W. and Tanaka, C. (1986) Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802 806. 8. Granier, F. (1988) Extraction of plant proteins for two-dimensional electrophoresis. Electrophoresis 9, 712 718. 9. Herbert, B. (1999) Advances in protein solubilisation for two-dimensional electrophoresis. Electrophoresis 20, 660 663. 10. Colas des Francs, C., Thiellement, H., and de Vienne, D. (1985) Analysis of leaf proteins by two-dimensional gel electrophoresis: protease action as exemplified by ribulose bisphosphate carboxylase/oxygenase degradation and procedure to avoid proteolysis during extraction. Plant Physiol. 78, 178 182.