Acquadro Alberto, Falvo Sara, Mila Silvia, Giuliano Albo Alessandra, Comino Cinzia, Moglia Andrea, Lanteri Sergio
DiVaPRA, Plant Genetics and Breeding, University of Torino, Grugliasco, Italy.
Electrophoresis. 2009 May;30(9):1594-602. doi: 10.1002/elps.200800549.
Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana.
在此，我们报告了对球茎甘蓝叶片蛋白质组的首次分析。测试了三种蛋白质提取方案，并建立了一种基于Mg/NP - 40的可重复方法。1,5 - 二磷酸核酮糖羧化酶/加氧酶（RuBisCO）是叶片中一种高度丰富的蛋白质，它的存在会掩盖共定位的、丰度较低的蛋白质。为了从样品中去除RuBisCO，从而提高斑点分辨率，精心设计了一种聚乙二醇分级分离方法。各种聚乙二醇级分的二维电泳图谱表明，分级分离程序成功地排除了大部分RuBisCO，从而能够检测到许多低丰度蛋白质。蛋白质免疫印迹分析能够证实通过聚乙二醇分级分离实现了RuBisCO含量的降低。总共检测到841个不同的蛋白质斑点，其中从二维电泳图谱的RuBisCO区域中选择的40个斑点通过质谱成功鉴定。这些蛋白质的许多同源物在拟南芥中也与RuBisCO共定位。
