Localization of organelle proteins by isotope tagging (LOPIT)
Localization of organelle proteins by isotope tagging (LOPIT)
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DOI：
10.1074/mcp.t400009-mcp200
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发表时间：
2004-11-01
影响因子：
7
通讯作者：
Lilley, KS
中科院分区：
文献类型：
--
作者：
Dunkley, TPJ;Watson, R;Lilley, KS
我们描述了一种用于确定膜蛋白亚细胞定位的蛋白质组学方法。细胞器通过自生成密度梯度使用离心部分分离。来自每个细胞器的蛋白质共破碎，因此在梯度中表现出相似的分布。蛋白质分布可以通过梯度级分的一系列成对比较来确定，使用可裂解的ICAT以使得能够通过MS相对定量蛋白质水平。使用多变量数据分析技术来确定新蛋白质的定位，以将它们的分布与已知存在于特定细胞器中的蛋白质的分布相匹配。使用这种方法，我们同时证明了定位的膜蛋白在内质网和高尔基体在拟南芥。细胞器蛋白质同位素标记定位技术是一种高通量蛋白质定位的新方法，适用于细胞器功能、蛋白质运输等研究领域。
We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through self-generating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.