Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants
Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants
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DOI：
10.1074/mcp.m600408-mcp200
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发表时间：
2007-04-01
影响因子：
7
通讯作者：
Shirasu, Ken
中科院分区：
文献类型：
--
作者：
Maor, Rudy;Jones, Alex;Shirasu, Ken
蛋白质与泛素结合，称为泛素化，是真核细胞中控制蛋白质丰度、定位和活性的关键调节机制。为了识别植物中泛素依赖的调控步骤，我们开发了一个强大的亲和纯化/鉴定系统的泛素化蛋白。利用GST标记的泛素结合域，我们进行了大规模的亲和纯化的泛素化蛋白质从拟南芥细胞悬浮培养。用SIDS-PAGE分离高分子量泛素化蛋白，然后用多维蛋白质鉴定技术（MudPIT）系统分析胰蛋白酶消化的样品。共鉴定了294种由GST标记的泛素结合结构域特异性结合的蛋白质。从这些，我们确定了85个泛素化的赖氨酸残基在56个蛋白质，确认富集的目标类蛋白质。我们的数据提供了植物中泛素化蛋白质组的第一个视图。我们还提供了证据表明，这种技术可以广泛应用于不同植物物种的蛋白质泛素化的研究。
Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SIDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (MudPIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.