Sugarcane proteomics: Establishment of a protein extraction method for 2-DE in stalk tissues and initiation of sugarcane proteome reference map
Sugarcane proteomics: Establishment of a protein extraction method for 2-DE in stalk tissues and initiation of sugarcane proteome reference map
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DOI：
10.1002/elps.200900779
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发表时间：
2010-06-01
期刊：
影响因子：
2.9
通讯作者：
Viswanathan, Rasappa
中科院分区：
文献类型：
--
作者：
Amalraj, Ramesh Sundar;Selvaraj, Nagarathinam;Viswanathan, Rasappa
甘蔗是一种重要的经济作物，因其茎而被种植，而糖是一种珍贵的商品，在人类营养中必不可少。甘蔗蛋白质组学还处于初级阶段，特别是在处理甘蔗茎组织时，到目前为止还没有研究。甘蔗茎组织的系统蛋白质组分析仍有待研究，其中茎组织以其刚性、纤维性以及氧化酶、酚类化合物和极高水平的碳水化合物的存在而闻名，因此使蛋白质提取变得复杂。在此，我们对甘蔗茎组织中蛋白质的五种不同提取方法进行了评价。这些方法包括：直接裂解缓冲液提取法、TCA/丙酮沉淀法、含硫脲(LBT)法、含虹膜LBT法、苯酚提取法。每种方法都进行了定量和定性的蛋白质分析。提取总蛋白的2-DE分析显示，不同方法之间的蛋白质图谱存在明显差异，这可能是由于它们的物理化学限制。从二维凝胶蛋白质图谱来看，TCA/丙酮沉淀-LBT和苯酚提取法效果较好。苯酚法显示蛋白质在二维凝胶上的等电点发生了漂移，这主要是通过蛋白质提取后使用二维清理试剂盒来克服的。在所测试的所有方法中，二维清除-苯酚法最适合产生高质量斑点和重现性。共筛选出了在LB法、LBT法、酚法和LBT法中普遍存在的30个蛋白质点和12个蛋白质点，分别进行了ELD-IT-TOF-MS/MS和NESI-LC-MS/MS分析，建立了甘蔗茎组织蛋白质组的参考图谱。共鉴定出36个非冗余蛋白。这是甘蔗茎蛋白质组分析的第一个基础性研究，将促进甘蔗蛋白质组研究的未知领域。
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 iris, 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.