ERK cascade scaffolds serve as docking platforms to coordinate the assembly of multiprotein complexes that contribute to the spatial and temporal control of ERK signaling. Given that protein–protein interactions are essential for scaffold function, determining the full repertoire of scaffold binding partners will likely provide new insight into the regulation and activities of the ERK cascade scaffolds. In this chapter, we describe methods to identify scaffold interacting proteins using a proteomics approach. This protocol is based on the affinity purification of scaffold complexes from tissue culture cells and utilizes mass spectrometry to identify the protein constituents of the complex.
