Protein Neighbors and Proximity Proteomics.
Protein Neighbors and Proximity Proteomics.
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DOI：
10.1074/mcp.r115.052902
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发表时间：
2015-11
期刊：
影响因子：
--
通讯作者：
Jackson AP
中科院分区：
文献类型：
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作者：
Rees JS;Li XW;Perrett S;Lilley KS;Jackson AP
在细胞内，蛋白质可以共组装成功能整合和空间限制的多组分复合物。通常，单个蛋白质之间的亲和力相对较弱，并且这些簇内的蛋白质可能仅与许多其他蛋白质邻居间接相互作用。这使得使用免疫沉淀或交联等方法进行蛋白质组学表征变得困难。最近，几个小组已经描述了使用酶催化的邻近标记试剂，其用小分子如荧光素或生物素共价标记靶蛋白的邻居。然后可以通过标准下拉方法分离修饰的蛋白质并通过质谱法鉴定。在这里，我们将描述这些技术以及它们的相似之处和差异。我们讨论了它们的应用，研究蛋白质组装，并提供了一种新的方式来表征细胞器蛋白质组。我们强调蛋白质组学定量和独立的目标验证在这样的实验中的重要性。此外，我们认为，有生物物理学和细胞生物学的原则，规定适当的酶催化的邻近标记方法，以解决特定的生物学问题的利益。
Within cells, proteins can co-assemble into functionally integrated and spatially restricted multicomponent complexes. Often, the affinities between individual proteins are relatively weak, and proteins within such clusters may interact only indirectly with many of their other protein neighbors. This makes proteomic characterization difficult using methods such as immunoprecipitation or cross-linking. Recently, several groups have described the use of enzyme-catalyzed proximity labeling reagents that covalently tag the neighbors of a targeted protein with a small molecule such as fluorescein or biotin. The modified proteins can then be isolated by standard pulldown methods and identified by mass spectrometry. Here we will describe the techniques as well as their similarities and differences. We discuss their applications both to study protein assemblies and to provide a new way for characterizing organelle proteomes. We stress the importance of proteomic quantitation and independent target validation in such experiments. Furthermore, we suggest that there are biophysical and cell-biological principles that dictate the appropriateness of enzyme-catalyzed proximity labeling methods to address particular biological questions of interest.