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Sugarcane proteomics: establishment of a protein extraction method for 2-DE in stalk tissues and initiation of sugarcane proteome reference map.

Electrophoresis (2010-06-22)
Ramesh Sundar Amalraj, Nagarathinam Selvaraj, Ganesh Kumar Veluswamy, Rahul Pathirickal Ramanujan, Raveendran Muthurajan, Malathi Palaniyandi, Ganesh Kumar Agrawal, Randeep Rakwal, Rasappa Viswanathan
ABSTRACT

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2-DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2-D gel protein profiles, TCA/acetone precipitation-LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2-D gel, which was mostly overcome by the use of 2-D cleanup kit after protein extraction. Among all the methods tested, 2-D cleanup-phenol method was found to be the most suitable for producing high number of good-quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD-IT-TOF-MS/MS and nESI-LC-MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Plant Fractionated Protein Extraction Kit, Suitable for any plant species or tissue
Sigma-Aldrich
Plant Hydrophobic Protein Extraction Kit, Suitable for any plant species or tissue
Sigma-Aldrich
Plant Total Protein Extraction Kit, Suitable for any plant species or tissue
Sigma-Aldrich
Plant Hydrophilic Protein Extraction Kit, Suitable for any plant species or tissue