Combining Phytate/Ca2+ Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis

doi:10.1016/S2095-3119(13)60344-5

Journal of Integrative Agriculture ›› 2013, Vol. 12 ›› Issue (7): 1123-1129.DOI: 10.1016/S2095-3119(13)60344-5

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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

收稿日期:2012-10-29 出版日期:2013-07-01 发布日期:2013-07-04
通讯作者: Correspondence ZHAO Hui-xian, Tel: +86-29-87092387, Fax: +86-29-87092262, E-mail: hxzhao212@yahoo.com.cn, hxzhao212@nwsuaf.edu.cn
基金资助:
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).

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

Received:2012-10-29 Online:2013-07-01 Published:2013-07-04
Contact: Correspondence ZHAO Hui-xian, Tel: +86-29-87092387, Fax: +86-29-87092262, E-mail: hxzhao212@yahoo.com.cn, hxzhao212@nwsuaf.edu.cn
Supported by:
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).

摘要/Abstract

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.

关键词: Triticum aestivum L. , Rubisco , low-abundance protein , phytate/Ca2+ , two-dimensional gel electrophoresis , plant leaf proteomics

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.

Key words: Triticum aestivum L. , Rubisco , low-abundance protein , phytate/Ca2+ , two-dimensional gel electrophoresis , plant leaf proteomics

Muhammad A R F Sultan, LIU Hui, CHENG Yu-Feng, ZHANG Pei-pei , ZHAO Hui-xian. Combining Phytate/Ca2+ Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis[J]. Journal of Integrative Agriculture, 2013, 12(7): 1123-1129.

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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

Combining Phytate/Ca2+ Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis

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