WP6: Affinity-based functional proteomics of signalling protein complexes

A key feature of protein binders is their ability to facilitate targeted analysis of protein complexes, pathways and networks. In order to derive full benefit from the unique collection of binders generated in the consortium, WP6 will explore applications of binders, ideally suited to target specific functions, in a functional proteomics approach. When coupled with mass spectrometry (MS), binders allow identification and characterisation of protein interactions from extracts or intact cells. With the aim of specifically validating binders against kinases, phosphatases and SH2 proteins involved in pathways for cell-cycle control, differentiation and cell survival, binder sets produced and validated in WP2 and WP3 will be used to analyse protein interaction networks in cells and lysates from selected organs and tumour tissue and following growth factor stimulation (HMGU, UTOV). Proteins will be identified and quantitated by MS and Western blotting (WB). In parallel, to analyse protein complexes in cells in situ, detection of proximity will be by DNA amplification in multiplexed proximity ligation (UU). Another key objective is to use affinity reagents for functional intracellular testing (intrabodies), including disruption of pathways/complexes, e.g. using nanobodies in cell-based screens (VIB). With one focus on the human kinome, binders will be used which target specific functions of kinases and kinase scaffolding proteins. Those interfering with the functions of PTMs, particularly phosphorylation, intrabodies can be expected to influence spatiotemporal dynamics of functional protein complexes by displacing components from their cognate intracellular location thereby generating potential sites for targeted disruption. RNAi arrays in combination with binders will also be used to cross-validate the effects obtained by disrupting complexes at the protein level in living cells (VTT).