15N-Metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells
15N-Metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells
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DOI：
10.1002/pmic.200600791
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发表时间：
2007-03-01
期刊：
影响因子：
3.4
通讯作者：
Thomine, Sebastien
中科院分区：
文献类型：
--
作者：
Lanquar, Viviane;Kuhn, Lauriane;Thomine, Sebastien
蛋白质组学研究的一个重要目标是对来自不同基因型、组织或生理条件的蛋白质组进行全局比较。迄今为止，这主要是通过2-DE分离蛋白质后的斑点强度的密度比较来实现的。然而，膜蛋白的物理化学性质阻止了2-DE的使用。在这里，我们描述了稳定同位素N-15在体内标记的使用，作为植物细胞中比较膜蛋白质组学研究的替代方法。我们证实了n -15代谢标记蛋白在拟南芥悬浮细胞中是可能的和有效的。N-14和N-15 MS信号的定量反映了分析样品中N-14和N-15蛋白的相对丰度。我们描述了使用n -15代谢标记来执行镉暴露后拟南芥细胞的部分比较分析。通过将我们的注意力集中在质膜蛋白上，我们能够自信地识别出与未暴露细胞相比表现出高达5倍调节的蛋白质。本研究提供了一个原理证明，n -15代谢标记是一个有用的技术比较膜蛋白质组研究。
An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope N-15 as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that N-15-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of N-14 versus N-15 MS signals reflects the relative abundance of N-14 and N-15 proteins in the sample analyzed. We describe the use of N-15-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that N-15-metabolic labeling is a useful technique for comparative membrane proteome studies.