Mapping protein 'interactomes' within membrane trafficking pathways: combining mass spectrometry and SILAC labelling with targeted tyramine tagging

真核细胞（即有核的细胞）含有丰富的内部膜结合区室。这些区室通常含有特殊的蛋白质，对细胞的健康有许多重要的作用。例如，我们开始认识到，这些途径中的蛋白质靶向错误是许多重要疾病的基础。现代细胞生物学活跃研究的一个主要领域是了解这些靶向事件是如何发生的。一般来说，蛋白质被选择性地靶向到不同的位置，因为它们与“外衣蛋白”相互作用，后者捕获并引导它们的目标到不同的内部区室。在这个过程中，外壳蛋白会短暂地聚集在细胞膜上。此外，许多被选择性靶向的蛋白质也出现在目标膜内的离散“斑块”中，因此局部分子邻居也可能影响这些相互作用。不幸的是，关于这些蛋白质复合物的性质仍然有很多不确定性。在试图描述它们的特性时，我们面临的许多技术问题是，许多与目标的相互作用只是短暂的，浓度相对较低，对目标的亲和力也较低。应该指出的是，这是一个普遍的问题，发生在细胞生物学的其他方面。例如，像胰岛素这样的激素会触发特定蛋白质在内部膜区室上的组装，同样，由于类似的原因，这些也很难表征。在这里，我们提出一种方法来解决这个普遍问题。它开发和扩展了在其他环境中成功使用的技术，但还没有在这种组合中使用。简单地说，我们将使用一种合适的蛋白质，这种蛋白质被一种叫做过氧化物酶的酶标记。这种酶可以将一种叫做酪胺的化学物质转化为一种非常不稳定的试剂，这种试剂只会“标记”紧邻该酶的分子。一个合适的蛋白质，标记过氧化物酶将被引入细胞。然后酪胺试剂将被添加到“标记”蛋白质和它的近邻。这些分子可以被识别并通过它们特定的“酪胺标签”纯化。一旦纯化，它们将被一种叫做质谱法的方法识别，这种方法可以成功地表征复杂混合物中的大量蛋白质。此外，我们建议包括专门改编的软件，以便轻松准确地分析使用该技术获得的任何数据。我们相信，我们的过程，我们称之为“靶向酪胺标记”，将在识别细胞内短暂相互作用的蛋白质-蛋白质伙伴的能力方面提供重大改进。

Eukaryotic cells (ie those cells with nuclei) contain a rich collection of internal membrane-bound compartments. These compartments often contain specialized proteins and serve many purposes vital for the well-being of the cell. For example, we are beginning to appreciate that protein mis-targeting within these pathways underlie a number of important diseases. A major area of active research within modern cell biology is to understand how these targeting events take place. In general terms, proteins are selectively targeted to different locations because they interact with 'coat proteins' that capture and direct their targets to different internal compartments. During this process, the coat proteins transiently assemble onto the internal membranes. Furthermore, many of the proteins that are selectively targeted also occur in discrete 'patches' within the targeted membrane themselves, and so the local molecular neighbours may also affect these interactions. Unfortunately, there is still much uncertainty concerning the nature of some of these protein complexes. Among the many technical problems we face when trying to characterize them is the fact that many interact with their targets only fleetingly and with relatively low concentrations and/or low affinity for targets. It should be noted that this is a general problem that occurs in other aspects of cell biology. For example, hormones such as insulin trigger the assembly of specific proteins onto internal membrane compartments and again these have been difficult to characterize for similar reasons. Here we propose a method to address this general problem. It develops and extends techniques that have been successfully used in other contexts, but not yet in this combination. Briefly, we will use a suitable protein labeled with an enzyme called peroxidase. This enzyme can convert a chemical called tyramine into a very unstable reagent that will only 'tag' molecules in the immediate vicinity of the enzyme. A suitable protein, labeled with peroxidase will be introduced into cells. Then tyramine reagent will be added to 'tag' both the protein and its immediate neighbours. These molecules can then be recognized and purified by their specific 'tyramine tag'. Once purified, they will be identified by a method called mass spectrometry that can successfully characterize large numbers of proteins in complex mixtures. In addition, we propose to include specifically adapted software that will enable the easy and accurate analysis of any data obtained with the technique. We believe that our process, that we call 'targeted tyramine tagging' will offer a significant improvement in the ability to identify transiently-interacting protein-protein partners within the cell.