Identification of ubiquitinated proteins in Arabidopsis
Identification of ubiquitinated proteins in Arabidopsis
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DOI：
10.1007/s11103-008-9358-9
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发表时间：
2008-09-01
影响因子：
5.1
通讯作者：
Del Pozo, Juan C.
中科院分区：
文献类型：
--
作者：
Manzano, Concepcion;Abraham, Zamira;Del Pozo, Juan C.
泛素（Ub）是一种小肽，在翻译后反应中共价附着在蛋白质上。泛素化是一种精确的调控系统，存在于所有真核生物中，调节蛋白质的稳定性、活性、定位和运输。泛素化涉及不同的酶活性，其中E3连接酶催化泛素标记靶标的最后一步招募。基因组分析表明，植物中泛素-蛋白酶体系统涉及大量蛋白质，约占总蛋白质的5%。与发现的大量泛素E3连接酶相反，很少有被泛素化调节的蛋白质被描述。为了解决这个问题，我们采用了一种新的蛋白质组学方法，旨在鉴定泛素修饰的蛋白质。这是基于使用附着在琼脂糖珠上的P62蛋白的泛素结合域（UBA）多肽对泛素化蛋白的亲和纯化和鉴定。这种p62琼脂糖基质能够特异性结合泛素化蛋白。结合蛋白经胰蛋白酶酶切，肽段经高效液相色谱分离，直接定位到MALDI靶上，通过MALDI-TOF/TOF离线耦合LC/MALDI-MS/MS进行分析。共鉴定出200个假定的泛素化蛋白。从这些研究中，我们发现一些假定的靶标已经在植物和其他生物中被描述为泛素化蛋白。此外，我们发现其中一些蛋白质确实在体内被泛素修饰。综上所述，我们已经证明这种方法对鉴定植物中的泛素化蛋白是有用的。
Ubiquitin (Ub) is a small peptide that is covalently attached to proteins in a posttranslational reaction. Ubiquitination is a precise regulatory system that is present in all eukaryotic organisms and regulates the stability, the activity, the localization and the transport of proteins. Ubiquitination involves different enzymatic activities, in which the E3 ligases catalyze the last step recruiting of the target for labelling with ubiquitin. Genomic analyses have shown that the ubiquitin-proteasome system involves a large number of proteins in plants, as approximately 5% of the total protein belongs to this pathway. In contrast to the high number of E3 ligases of ubiquitin identified, very few proteins regulated by ubiquitination have been described. To solve this, we have undertaken a new proteomic approach aimed to identify proteins modified with ubiquitin. This is based on affinity purification and identification for ubiquitinated proteins using the ubiquitin binding domain (UBA) polypeptide of the P62 protein attached to agarose beads. This P62-agarose matrix is capable of specifically binding ubiquitinated proteins. These bound proteins were digested with trypsin and the peptides separated by HPLC chromatography, spotted directly onto a MALDI target and analyzed by MALDI-TOF/TOF off-line coupled LC/MALDI-MS/MS. A total of 200 putative ubiquitinated proteins were identified. From these we found that several of the putative targets were already described in plants, as well as in other organisms, as ubiquitinated proteins. In addition, we have found that some of these proteins were indeed modified with ubiquitin in vivo. Taken together, we have shown that this approach is useful for identifying ubiquitinated protein in plants.