Dunkley Tom P J, Hester Svenja, Shadforth Ian P, Runions John, Weimar Thilo, Hanton Sally L, Griffin Julian L, Bessant Conrad, Brandizzi Federica, Hawes Chris, Watson Rod B, Dupree Paul, Lilley Kathryn S
Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom.
Proc Natl Acad Sci U S A. 2006 Apr 25;103(17):6518-23. doi: 10.1073/pnas.0506958103. Epub 2006 Apr 17.
A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.
分泌途径研究中的一项具有挑战性的任务是鉴定新蛋白质并确定其定位，以增进我们对不同细胞器功能的理解。以往对内膜系统的蛋白质组学研究受到污染蛋白的阻碍，使得无法将蛋白质分配到细胞器。在此，我们利用同位素标记技术对细胞器蛋白质进行定位，结合用于相对和绝对定量的同位素标签以及二维液相色谱，以便将蛋白质同时分配到多个亚细胞区室。通过这种方法，确定了来自拟南芥的689种蛋白质的密度梯度分布，从而能够将527种蛋白质可靠地同时定位到内质网、高尔基体、液泡膜、质膜或线粒体和质体。这种对内膜成分的平行分析使得能够确定蛋白质的稳态分布。因此，真正的细胞器驻留蛋白已与污染蛋白以及通过分泌途径转运的蛋白区分开来。
