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| Combining Phytate/Ca2+ Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis |
| Muhammad A R F Sultan, LIU Hui, CHENG Yu-Feng, ZHANG Pei-pei , ZHAO Hui-xian |
| State Key Laboratory of Crop Stress Biology for Arid Areas, Ministry of Science and Technology/College of Life Sciences, Northwest A&F University, Yangling 712100, P.R.China |
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摘要 Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca2+ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.
Abstract Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca2+ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.
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Received: 29 October 2012
Accepted:
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| Fund: This research was supported by the National Natural Science Foundation of China (30871578) and the Key Project of National Plant Transgenic Genes of China (2008ZX08002004, 2011ZX08002004). |
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Corresponding Authors:
Correspondence ZHAO Hui-xian, Tel: +86-29-87092387, Fax: +86-29-87092262, E-mail: hxzhao212@yahoo.com.cn, hxzhao212@nwsuaf.edu.cn
E-mail: hxzhao212@yahoo.com.cn
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Cite this article:
Muhammad A R F Sultan, LIU Hui, CHENG Yu-Feng, ZHANG Pei-pei , ZHAO Hui-xian.
2013.
Combining Phytate/Ca2+ Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis. Journal of Integrative Agriculture, 12(7): 1123-1129.
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| [1]Bradford M M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizingthe principle of protein-dye binding. AnalyticalBiochemistry, 72, 248-254[2]Chen Q, Zhang M D, Shen S H. 2011. Comparison of proteinextraction methods suitable for proteomics analysis inseedling roots of Jerusalem artichoke under salt (NaCl)stress. African Journal of Biotechnology, 10, 7650-7657[3]Cheryan M. 1980. Phytic acid interactions in food system.Critical Reviews in Food Science and Nutrition, 13,297-335[4]Cho J H, Hwang H, Cho M H, Kwon Y K, Jeon J S, Bhoo SH, Hahn T R. 2008. The effect of DTT in proteinpreparations for proteomic analysis: removal of a highlyabundant plant enzyme, ribulose bisphosphatecarboxylase/oxygenase. Journal of Plant Biology, 51,297-301[5]Corthalis G L, Wasinger V C, Hochstrasser D F, Sanchez JC. 2000. The dynamic range of protein expression: achallenge for proteomic research. Electrophoresis, 21,1104-1115[6]Damerval C, Vienne D D, Zivy M, Thiellement H. 1986.Technical improvements in two-dimensionalelectrophoresis increase the level of genetic variationdetected in wheat-seedling proteins. Electrophoresis,7, 52-54[7]Görg A, Weiss W, Dunn M J. 2004. Current two-dimensionalelectrophoresis technology for proteomics. Proteomics,4, 3665-3685[8]Gomez-Vidal S, Tena M, Lopez-Llorca L, Salinas J. 2008.Protein extraction from Phoenix dactylifera L. leaves, arecalcitrant material, for two-dimensionalelectrophoresis. Electrophoresis, 29, 448-456[9]Hurkman W J, Tanaka C K. 1986. Solubilization of plantmembrane proteins for analysis by two-dimensional gelelectrophoresis. Plant Physiology, 81, 802-806[10]Herman E M, Helm R M, Jung R, Kinney A J. 2003. Geneticmodification removes an immunodominant allergen fromsoybean. Plant Physiology, 132, 36-43[11]Krishnan H B, Natarajan S S. 2009. A rapid method fordepletion of Rubisco from soybean (Glycine max) leaffor proteomic analysis of lower abundance proteins.Phytochemistry, 70, 1958-1964[12]Laemmli U K. 1970. Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature,227, 680-685[13]Lakshman D K, Natarajan S S, Lakshman S, Garrett W M,Dhar A K. 2008. Optimized protein extraction methodsfor proteomic analysis of Rhizoctonia solani.Mycologia, 100, 867-875[14]McKinney M M, Parkinson A. 1987. A simple, nonchromatographicprocedure to purify immunoglobulinsfrom serum and ascites fluid. Journal of ImmunologicalMethods, 2, 271-278[15]Nolan K B, Duffin P A, McWeeny D J. 1987. Effects ofphytate on mineral bioavailability. In vitro studies onMg2+, Ca2+, Fe3+, Cu2+ and Zn2+ (also Cd2+) solubilities inthe presence of phytate. Journal of the Science of Foodand Agriculture, 40, 79-85[16]O’Farrel P H. 1975. High resolution two-dimensionalelectrophoresis of proteins. The Journal of BiologicalChemistry, 250, 4007-4021[17]Rogers A, Ellsworth D S, Humphries S W. 2001. Possibleexplanation of the disparity between the in vitro and invivo measurements of Rubisco activity - A study inloblolly pine grown in elevated pCO2. Journal ofExperimental Botany, 52, 1555-1561[18]Saravanan R S, Rose J K. 2004. A critical evaluation ofsample extraction techniques for enhanced proteomicanalysis of recalcitrant plant tissues. Proteomics, 4,2522-2532[19]Wang X, Li X, Deng X, Han H, Shi W, Li Y. 2007. A proteinextraction method compatible with proteomic analysisfor the euhalophyte Salicornia europaea. Electrophorsis,28, 3976-3987[20]Xi J, Wang X, Li S, Zhou X, Yue L, Fan J, Hao D. 2006.Polyethylene glycol fractionation improved detectionof low-abundant proteins by two dimensional electrophoresisanalysis of plant proteome. Phytochemistry, 67,2341-2348[21]Xie H, Pan S, Liu S, Ye K, Huo K. 2007. A novel method ofprotein extraction from perennial Bupleurum root for2-DE. Electrophoresis, 28, 871-875[22]Zhang R, Liu H, Zhao H, Hu S. 2010. Comparison of twoprotein extraction methods for proteomic analysis ofchlorophyll-deficient mutants in Brassica juncea L.Progress in Biochemistry and Biophysics, 37, 1025-1032 |
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