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

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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.