Nat. Biotechnol. 29, 653–658 (2011)

Adaptor proteins such as GRB2 are hubs for protein-protein interactions (PPIs) that convey signals from cell surface receptors to downstream effectors. Understanding the dynamics of these signaling networks in stimulated cells remains challenging. Bisson et al. now report affinity purification–selected reaction monitoring (AP-SRM) as a new MS–based approach to evaluate context- and time-specific PPIs with GRB2 after growth factor stimulation. To validate the method, the authors show that the function of SH2 or SH3 is necessary to detect proteins known to interact with GRB2 via these domains. The authors applied AP-SRM to quantify the interaction between GRB2 and 90 signaling proteins upon activation with epidermal growth factor in a time-dependent manner, identifying three groups of proteins that bind GRB2 with distinct kinetics. Finally, the authors quantified PPIs upon activation with one of six different growth factors to identify the core GRB2 machinery, which includes proteins that interact with GRB2 under multiple conditions as well as growth factor–specific PPI networks. Compared to other MS strategies, AP-SRM is highly sensitive, reproducible and quantitative, and it does not require in vivo labeling. Thus, AP-SRM provides a robust alternative for hypothesis-driven investigations to quantitatively measure the dynamics of PPI networks.