Protocol for Phosphoprotein Enrichment in Rice (Oryza sativa L.) —Phenol Extraction Combined with Immobilized Metal Affinity Chromatography
DENG Xiang-Xiong1）, ZHANG Xiao-Qin1）, HU Jiang2）, XU Xiang-Bin1）, GUO Long-Biao2）, CHEN Hui-Nan1）, YING Qi-Cai1）, WANG Hui-Zhong1）, XUE Da-Wei1)*
1)College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou310036, China;
2) State Key Laboratory of Rice Biology, National Rice Research Institute of China,Hangzhou310006, China
Abstract：Phosphoproteins play very important roles in plant signal transduction and stress responses. Phosphoproteomics has become a big concern for proteomic researchers. Total protein was extracted from rice (Oryza sativa L. Japonica nipponbare) leaves via phenol extraction in this study. Protein mass obtained is up to 3.5% in fresh rice leaves; phosphoprotein was enriched by an immobilized metal affinity chromatography column, which takes a proportion of 6.4% in the total protein. Protein profiles of washing solution, elution solutions and others were approximately exhibited by SDS-PAGE. Based on the results, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for total proteins, proteins from peak fraction, nonpeak fractions and purified phosphoprotein after enrichment were carried out to better understand the variance among these samples on two dimensional gels. The protocol and procedure described here could dissociate as more as 856 spots per polyacrylamide gel of 7 cm. It is a highly effective way to separate and detecte phosphoproteins.
邓湘雄，张晓勤，胡江，徐祥彬，郭龙彪，陈慧男，应奇才，王慧中，薛大伟. 一种适用于双向电泳的水稻磷酸化蛋白富集方法——酚提取法结合固相金属离子亲和层析法[J]. 中国生物化学与分子生物学报, 2012, 28(11): 1057-1063.
DENG Xiang-Xiong1）, ZHANG Xiao-Qin, HU Jiang, XU Xiang-Bin, GUO Long-Biao, CHEN Hui-Nan, YING Qi-Cai, WANG Hui-Zhong, XUE Da-Wei. Protocol for Phosphoprotein Enrichment in Rice (Oryza sativa L.) —Phenol Extraction Combined with Immobilized Metal Affinity Chromatography. Chinese Journal of Biochemistry and Molecular Biol, 2012, 28(11): 1057-1063.
Weiss W, Weiland F, Görg A. Protein detection and quantitation technologies for gel-based proteome analysis [J]. Methods Mol Biol,2009, 564：59-82
Zolnierowicz S, Bollen M. Protein phosphorylation and protein phosphatases. De Panne, Belgium, September 19-24,1999 [J]. EMBO J,2000, 19(4)：483-488
Delom F, Chevet E. Phosphoprotein analysis: from proteins to proteomes [J]. Proteome Sci,2006, 4：15
刘金凤,王京兰,钱小红,等. 翻译后修饰蛋白质组学研究的技术策略[J]. 中国生物化学与分子生物学报, (Liu Jin-Feng, Wang Jing-Lan, Qian Xiao-Hong, et al. Technique strategies of modification-specific proteomics study[J]. Chin J Biochem Mol Biol), 2007, 23(2)：93-100
Paradela A, Albar J P. Advances in the analysis of protein phosphorylation [J]. J Proteome Res, 2008, 7(5)：1809-1818
Raggiaschi R, Lorenzetto C, Diodato E, et al. Detection of phosphorylation patterns in rat cortical neurons by combining phosphatase treatment and DIGE technology [J]. Proteomics,2006,6(3)：748-756
de la Fuente van Bentem S, Hirt H. Using phosphoproteomics to reveal signalling dynamics in plants [J]. Trends Plant Sci, 2007,12(9)：404-411
Nagano K, Shinkawa T, Yabuki N, et al. Integration of proteomic analyses to monitor the activity status of phosphorylation signaling [J]. J Proteomics, 2011, 74(3)：319-326
Agrawal G K, Jwa N S, Rakwal R. Rice proteomics: ending phase I and the beginning of phase II [J]. Proteomics, 2009, 9(4)：935-963
Yoshida S, Forna DA, Cock JH, et al. Laboratory manual for physiological studies of rice. International Rice Research Institute, Los Banos [M], Philippines, 1976, 62-63
Carpentier S C, Witters E, Laukens K, et al. Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis [J]. Proteomics, 2005, 5(10)：2497-2507
O’Farrell P H. High resolution two-dimensional electrophoresis of protein [J]. J Biol Chem, 1975, 250 (10)：4007-4021
Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Anal Biochem,1976,72：248-254
Görg A, Postel W, Westermeier R, et al. Gel gradient electrophoresis, isoelectric focusing and two- dimensional techniques in horizontal, ultrathin polyacrylamide layers[J]. J Biochem Biophys Methods [J].1980,3(5)：273-284
Wilkins M R, Sanchez J C, Williams K L, et al. Current challenges and future applications for protein maps and posttranslational vector maps in proteome projects [J]. Electrophoresis,1996,17(5)：830-838
Damerval C, Vienne D D, Zivy M, et al. Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins [J]. Electrophoresis,1986,7(1)：52-54
Méchin V, Damerval C, Zivy M. Total protein extraction with TCA-acetone [J]. Methods Mol Biol, 2007, 355：1-8
Yang P, Li X, Wang X, et al. Proteomic analysis of rice (Oryza sativa) seeds during germination [J]. Proteomics,2007,7(18)：3358-3368
Zhang X, Ye J, Jensen O N, et al. Highly efficient phosphopeptides enrichment by calcium phosphate precipitation combined with subsequent IMAC enrichment [J]. Mol Cell Proteomics,2007,6(11)：2032- 2042
Shi F, Takasaki H, Komatsu S. Quantitative analysis of auxin-regulated proteins from basal part of leaf sheaths in rice by two-dimensional difference gel electrophoresis [J]. Phytochemistry,2008,69(3)：637-646
Komatsu S, Yamada E, Furukawa K. Cold stress changes the concanavalin A-positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths [J]. Amino Acids,2008,36(1)：115-123
Hashimoto M, Komatsu S. Proteomic analysis of rice seedlings during cold stress [J]. Proteomics, 2007,7(8)：1293-1302
Li G, Nallamilli B R, Tan F, et al. Removal of high-abundance proteins for nuclear subproteome studies in rice (Oryza sativa) endosperm [J]. Electrophoresis,2008,29(3)：604-617
Miché L, Battistoni F, Gemmer S, et al. Upregulation of jasmonate-inducible defense proteins and differential colonization of roots of Oryza sativa cultivars with the endophyte Azoarcus sp [J]. Mol Plant Microbe Interact,2006,19(5)：502-511
Chitteti B R, Peng Z. Proteome and phosphoproteome differential expression under salinity stress in rice (Oryza sativa) roots [J]. J Proteome Res,2007,6(5)：1718-1727
Ahsan N, Lee D G, Lee S H, et al. Excess copper induced physiological and proteomic changes in germinating rice seeds [J]. Chemosphere,2007,67(6) ：1182-1193
Ahsan N, Lee S H, Lee D G, et al. Physiological and protein profiles alternation of germinating rice seedlings exposed to acute cadmium toxicity [J]. C R Biol,2007,330(10)：735-746
Lee DG, Ahsan N, Lee SH, et al. An approach to identify cold-induced low-abundant proteins in rice leaf [J]. C R Biol,2007,330(3)：215-225
Yin L, Tao Y, Zhao K, et al. Proteomic and transcriptomic analysis of rice mature seed-derived callus differentiation [J]. Proteomics,2007,7(5)：755-768
Fukuda T, Saito A, Wasaki J, et al. Metabolic alterations proposed by proteome in rice roots grown under low P and high Al concentration under low pH [J]. Plant Sci,2007,172(6)：1157-1165
Zhang L L, Feng R J, Zhang Y D. Evaluation of different methods of protein extraction and identification of differentially expressed proteins upon ethylene-induced early-ripening in banana peels [J]. J Sci Food Agric,2012,92(10):2106-2115
Faurobert M, Pelpoir E, Chaïb J. Phenol Extraction of Proteins for Proteomic Studies of Recalcitrant Plant Tissues [J]. Methods Mol Biol,2007,355：9-14
Saravanan R S, Rose J K. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues [J]. Proteomics,2004,4(9)：2522-2532
