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DOI 10.1002/pmic.200600493
Proteomics 2006, 6, 6494–6497
TECHNICAL BRIEF
Potato tuber proteomics: Comparison of two complementary extraction methods designed for 2-DE of acidic proteins Pierre Delaplace1, Froukje van der Wal2, Jean-Franc¸ois Dierick3, Jan H. G. Cordewener2, Marie-Laure Fauconnier1, Patrick du Jardin1 and Antoine H. P. America2 1
Plant Biology Unit, Gembloux Agricultural University, Gembloux, Belgium BU Bioscience, Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands 3 BioVallée, Proteomics Unit, Charleroi, Belgium 2
Two protein extraction procedures were tested in order to remove interfering compounds prior to 2-DE of potato tubers. These methods using SDS lysis buffer and phenol-phase extraction were compared regarding the quality of the resulting 2-D gel. While the resolution of SDS extracts on semipreparative gels seems better, both methods lead to similar extraction yields and total number of spots. The procedures are complementary regarding the Mr range of preferentially extracted proteins.
Received: July 7, 2006 Revised: July 31, 2006 Accepted: August 21, 2006
Keywords: Extraction method / Phenol / Plant proteomics / SDS / Two-dimensional gel electrophoresis
In spite of new gel-free protein separation technologies (e.g., multidimensional LC, stable isotope labeling, protein or antibody arrays), currently, 2-DE with IPGs remains the only technique that can be routinely applied for expression profiling of large sets of complex protein mixtures [1]. For 2-DE, the choice of a sample preparation protocol is the critical factor influencing IEF which in turn affects the gel pattern quality. This is especially true when dealing with plant tissues because of the inherent characteristics of this matrix: low protein/volume content, high concentration of interfering compounds such as phenolics, pigments, proteolytic, oxidative enzymes, and carbohydrates (like starch). Early researches using 2-DE-compatible potato tuber extraction methods were either based on denaturating Correspondence: Dr. Pierre Delaplace, Plant Biology Unit, Gembloux Agricultural University, Avenue de la Faculté d’Agronomie 2A, B5030 Gembloux, Belgium E-mail:
[email protected] Fax: +32-81-622460 Abbreviations: FW, fresh weight; RT, room temperature
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
extraction in lysis buffer without precipitation [2] or TCA/ acetone precipitation techniques [3]. This precipitation step is now regarded as an absolute necessity when dealing with recalcitrant tissues in order to obtain high quality 2-DE profiling [4]. Recent studies aiming at evaluating several TCA/ acetone- or phenol-based extraction protocols [4–8] for proteomic analysis of recalcitrant plant tissues concluded that both types of protocols proved useful as standard methods. The phenol extraction method apparently gave a higher protein yield and typically higher quality gels, particularly with extracts from tissue containing high levels of soluble polysaccharides [4, 5, 7]. Here we report the comparison of a phenol extraction method derived from the protocol of Hurkman and Tanaka [9] versus an SDS-based extraction protocol developed at Plant Research International (Wageningen, The Netherlands) and modified from [10, 11] (Fig. 1). Potato (Solanum tuberosum L. cv. Désirée) tuber tissues (stored at 47C, 90% relative humidity) were frozen in liquid nitrogen and powdered in an IKA A10 mill (IKA-Werke, Staufen, Germany) prior to storage at 2807C. www.proteomics-journal.com
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Figure 1. Workflow comparison of Phenol- and SDS-based extraction methods. The @*@ indicates optional steps. The optional DIGE labeling buffer is 7 M urea, 2 M thiourea, 4% w/v CHAPS, 30 mM Tris, pH 8.5. O/N: overnight; RT: room temperature.
Phenol-extracted proteins were obtained as follows. Approximately 1 g of potato tuber powder (fresh weight, FW) was homogenized and incubated on ice for 10 min with 4 mL of extraction buffer (0.7 M sucrose, 50 mM EDTA, 0.1 M KCl, 10 mM thiourea, 0.5 M Tris, pH 7.5 with 2 mM PMSF and 50 mM DTT added sequentially). The homogenate was centrifuged (15 min, 13 0006g, 47C) and the supernatant was subsequently extracted (by vortexing at 1800 rpm) for 10 min with 5 mL of pH 8.0 buffered phenol at room temperature (RT). After centrifugation (10 min, 60006g, RT), the phenol phase was re-extracted with 5 mL of extraction buffer during 10 min (RT) and centrifuged again using the same parameters. The buffer phase was removed. The proteins contained in the phenol phase were precipitated overnight at 2207C by the addition of 20 mL of 0.1 M ammonium acetate in methanol and then centrifuged at 20 0006g for 20 min at 47C. The pellet was washed twice with 4 mL of 0.1 M cold ammonium acetate in methanol and once with 10 mM DTT cold acetone. The washed pellet was air-dried for 30 min at RT and then © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
solubilized in 600 mL of a rehydration buffer modified from [12, 13] (5 M urea, 2 M thiourea, 2% w/v CHAPS, 2% w/v 3-(4-heptyl)phenyl-3-hydroxy-propyl-dimethylammonio-propanesulfonate (C7BzO), 20 mM DTT, 5 mM tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl)) during 45 min at RT before storage at 2807C. The rehydration buffer volume was added in one step to thoroughly wet the inner walls of a 50 mL tube and dissolve the protein pellet. SDS-extracted proteins were obtained after the following steps. Approximately 1 g of potato tuber powder was repeatedly mixed and incubated at 657C for 10 min with 2 mL of SDS lysis buffer (4% w/v SDS, 5% w/v sucrose, 10% w/v poly(vinylpolypyrrolidone), 0.3% w/v DTT, 20 mM sodium phosphate, pH 7.0) preheated to 657C. The homogenate was then cooled on ice for 15 min before centrifugation (15 min, 15 0006g, 47C). An additional extraction step could be performed on the residue before the following steps (optional). The resulting supernatant(s) was/were centrifuged again using the same parameters before overnight precipitation www.proteomics-journal.com
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using 8 mL of 10 mM DTT cold acetone and subsequent centrifugation (20 min, 20 0006g, 47C). The protein pellet was washed twice with 10 mM DTT cold acetone. The washed pellet was then air-dried for 30 min at RT and then solubilized in 300 mL of the same rehydration buffer as for the phenol-extracted samples during 45 min at RT before rinsing the 15 mL tube inner walls with 200 mL of fresh buffer during 30 min. Both fractions were pooled and stored at 2807C. The protein concentration was determined using the RC/ DC protein assay from BioRad (Hercules, CA, USA). The IEF was performed using 24 cm pH 4–7 IPG strips (GE Healthcare, Little Chalfont, UK). The IPG strips were rehydrated overnight with 450 mg of total proteins diluted in rehydration buffer (see above) complemented with 1.2 mL of IPG buffer, pH 4–7 (GE Healthcare) and 1.2 mL of IPG buffer pH 3–10 (GE Healthcare) to reach a final volume of 450 mL. After rehydration, the focusing was performed on the IPGphor (GE Healthcare) using the following conditions: 30 V during 1 h, 300 V during 3 h, gradient to 1000 V in 6 h, gradient to 8000 V in 3 h, 8000 V until 100 000 Vh. Prior to second dimension, the strips were equilibrated for 15 min in 10 mL equilibration buffer (6 M urea, 30% glycerol, 2% SDS, 50 mM Tris pH 8.8) containing 1% w/v DTT and subsequently for 15 min in 10 mL equilibration buffer containing 2.5% w/v iodoacetamide. The separation in the second dimension was realized on the Ettan DALTsix System (GE Healthcare) with lab cast 1 mm SDS polyacrylamide gels (12.5%): 1 h 2 W/gel, 3 h30 100 W total. Acrylamide was purchased from BioRad. The gels were individually stained with 200 mL SYPRO Ruby fluorescent stain (BioRad) according to the manufacturer’s instructions. Gel images were acquired with the Typhoon 9200 (GE Healthcare) using an excitation wavelength of 532 nm and an emission wavelength filter of 610 nm. Images produced from three independent extracts for each extraction method were analyzed using the Progenesis PG220 v2006 software (Nonlinear Dynamics, Newcastle upon Tyne, UK). Spot detection was automatically performed with minimal manual editing to eliminate automatic detection errors (e.g., due to minor streaking, gel side effects, or staining speckles). Phenol extraction yields are 4.7 6 1.2 mg protein/mg FW (mean of triplicate 6 SD). Single SDS extraction yields are lower (2.5 6 0.4 mg protein/mg FW) but reach comparable values (4.3 6 0.7 mg protein/mg FW) with the additional extraction step. This extraction step may be regarded as optional since no modification in the protein profile was observed using one or two extraction steps of the tuber powder either on SDS-PAGE or 2-DE 7 cm-wide gels (data not shown). The experimental conditions used to compare both extraction methods are typical for semipreparative gels regarding the protein loading (450 mg). The pI range (4–7) ensures a good horizontal resolution since most of the extracted proteins are neutral to acidic [4, 8]. Total spot © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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numbers for phenol and SDS extracts were, respectively, 1572 6 124 and 1482 6 109 (means of triplicate gels 6 SD). This difference cannot be considered as statistically significant (Student’s t-test, p = 0.05). The number of common spots (e.g., patatin isoforms) is low and represents only a small fraction of the total spot number. Concerning the Mr range of the resolved proteins, phenol extracts exhibited more proteins with an Mr.40 kDa (955 6 86, 60.8% of the total proteins) than SDS extracts (501 6 92, 33.8% of the total proteins). This evaluation is based on the position of the patatin isoforms (P15478, MW 42 kDa, see [8]) on the gels (Fig. 2). On the contrary, SDS extraction method exhibited more lower MW proteins (,40 kDa, 981 6 74, 66.2% of total proteins) compared to phenol extracts (617 6 54, 39.2% of total proteins). It is unlikely that this MW range difference could be caused by a different of proteolytic breakdown since the SDS-extracted proteases are supposed to be irreversibly inactivated [10, 11]. Residual polysaccharides potentially present in SDS-extracted samples could narrow the gel pores and create a bias toward smaller proteins that are able to enter the gel. As far as the overall gel quality is concerned, phenol extracts display more horizontal streaking on gels than SDS extracts. This characteristic has not been emphasized in recent publications dealing with potato leaf- or tuber-proteins 2-DE based on phenol extraction [4, 8]. However, in such studies, the protein load was typically lower (between 40 and 300 mg total proteins/gel) than the one we used in our experimentation. The main reasons generating horizontal streaking could be under- or overfocusing (over 100 000 Vh) in the first dimension, protein overloading, sample preparation problems, poor protein solubilization, or endoosmotic flow [14]. Gels protein profiles (7 cm wide protein load: 20 mg) focused using 12 000 Vh during IEF presented the same tendency (data not shown). Even if we cannot exclude an overfocusing problem totally, the most plausible explanation seems related to inherent sample characteristics leading to overloading sensibility. In our preparative conditions, SDS extracts could thus be considered more tolerant to higher protein loads. This characteristic can ease the transition between analytical and preparative gels, hence facilitating protein identification by MS. In conclusion, we have presented and compared two complementary extraction methods concerning the Mr range of extracted proteins. The SDS extraction method will allow a higher protein loading per gel and is easier to perform (less extraction and washing steps), which can potentially improve the reproducibility between independent extracts. The use of SDS anionic detergent could also improve the membrane [1, 15]- and starch granule-associated protein’s solubilization. The detailed characterization (post-translational glycosylation pattern, hydrophobicity, etc.) of the proteins that are preferentially extracted with both methods is complex (see concluding remarks of [4]) and remains to be investigated. www.proteomics-journal.com
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Figure 2. Evaluation of the phenol-based (a) and SDS-based (b) extraction methods for potato tubers. Total numbers of spots are, respectively, 1572 6 124 and 1482 6 109 (mean values of triplicate gels 6 SD) for the phenol-based and the SDS-based extraction methods. Arrows indicate 42kDa patatin isoforms that were identified in [8]. Separation was performed on 24cm strips (pI 4–7).
This work was supported by short term grants from the Fond National de la Recherche Scientifique (Belgium) and the Netherlands Proteomics Centre Hotel facility (Wageningen, the Netherlands). The work of Jean-Franc¸ois Dierick is funded by the Région Wallonne (EP1A12030000082) FEDER BioVallée PROJECT.
[6] Koistinen, K. M., Hassinen, V. H., Gynther, P. A. M., Lehesranta, S. J. et al., New Phyt. 2002, 155, 381–391. [7] Saravanan, R. S., Rose, J. K. C., Proteomics 2004, 4, 2522– 2532. [8] Lehesranta, S. J., Davies, H. V., Shepherd, L. V. T., Nunan, N. et al., Plant Physiol. 2005, 138, 1690–1699. [9] Hurkman, W. J., Tanaka, C. K., Plant Physiol. 1986, 81, 802– 806.
References [1] Görg, A., Weiss, W., Dunn, M. J., Proteomics 2004, 4, 3665– 3685. [2] Espen, L., Morgutti, S., Cocucci, S. M., Potato Res. 1999, 42, 203–214. [3] Désiré, S., Couillerot, J.-P., Hilbert, J.-L., Vasseur, J., Plant Physiol. Biochem. 1995, 33, 479–487.
[10] Harrison, P. A., Black, C. C., Plant Physiol. 1982, 70, 1359– 1366. [11] Colas des Francs, C., Thiellement, H., de Vienne, D., Plant Physiol. 1985, 78, 178–182. [12] Rabilloud, T., Blisnick, T., Heller, M., Luche, S. et al., Electrophoresis 1999, 20, 3603–3610. [13] Méchin, V., Consoli, L., Le Guilloux, M., Damerval, C., Proteomics 2003, 3, 1299–1302.
[4] Carpentier, S. C., Witters, E., Laukens, K., Deckers, P. et al., Proteomics 2005, 5, 2497–2507.
[14] Berkelman, T., Stenstedt, T., 2-D Electrophoresis Using Immobilized pH Gradients: Principles and Methods, Amersham Biosciences, Upssala, Sweden 1998.
[5] Damerval, C., de Vienne, D., Zivy, M., Thiellement, H., Electrophoresis 1986, 7, 52–54.
[15] Song, J., Braun, G., Bevis, E., Doncaster, K., Electrophoresis 2006, 27, 3144–3151.
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