Proteomics Approaches For Studing Retrograde Transport
Analyzing The Dynamic Protein Traffic
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Proteomics is a powerful technique for the large-scale identification of proteins. A number of proteomics approaches have been developed to study protein localization at steady state. Here we report a novel dynamic proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans-Golgi network. This strategy is based on the covalent modification of the plasma membrane proteome with a benzylguanine derivative. Only benzylguanine-tagged plasma membrane proteins that are transported via the retrograde route can covalently couple to a TGN-localized SNAP-tag fu...
Proteomics is a powerful technique for the large-scale identification of proteins. A number of proteomics approaches have been developed to study protein localization at steady state. Here we report a novel dynamic proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans-Golgi network. This strategy is based on the covalent modification of the plasma membrane proteome with a benzylguanine derivative. Only benzylguanine-tagged plasma membrane proteins that are transported via the retrograde route can covalently couple to a TGN-localized SNAP-tag fusion protein. The approach was validated step-by-step using a well explored retrograde cargo protein, the B-subunit of Shiga toxin. It was then extended in an unbiased manner to the proteomics format. This allowed identifying an endogenous protein that was one of the first retrograde cargo proteins to be described in the past. In conclusion, we have pioneered a dynamic proteomics approach that complements traditional approaches in the study of protein trafficking. Moreover, the concept that is described here is of generic nature and may be extended to other endocytic pathways.
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