Subunit architecture of multimeric complexes isolated directly from cells
Subunit architecture of multimeric complexes isolated directly from cells
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DOI：
10.1038/sj.embor.7400702
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发表时间：
2006-06-01
期刊：
影响因子：
7.7
通讯作者：
Robinson, Carol V.
中科院分区：
文献类型：
--
作者：
Hernandez, Helena;Dziembowski, Andrzej;Robinson, Carol V.
在纯化策略方面的最新进展，以及基于质谱仪(MS)的蛋白质组学，已经确定了大量体内蛋白质复合体，并表明还有许多其他蛋白质复合体的存在。然而，标准的蛋白质组学技术无法描述这些组件的整体化学计量、亚单位相互作用和组织，因为许多组件是异质的，存在于相对较低的细胞丰度，并且经常难以分离。我们结合了两种现有的方法来应对这些挑战：串联亲和纯化以分离足够数量的高纯度天然化合物，以及MS对完整的组装和子复合体进行鉴定以确定其结构组织。我们用来自酿酒酵母的两个蛋白质组件(清道夫解帽和核帽结合复合体)优化了我们的方案，建立了亚基化学计量比，并鉴定了亚化学计量结合。然后我们以酵母外切体为目标，这是一种具有10个不同亚基的核酸酶，并发现通过产生亚复合体，可以得到三维相互作用图，展示了我们的方法对于大型、异质细胞复合体的实用性。
Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae ( scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.