Title: Quantitative Mass Spectrometry Analysis of Signalling Dynamics Using Selected Reaction Monitoring: a Case for the Adaptor Protein GRB2 - Nature Biotechnology (2011)

Authors: Nicolas Bisson, D. Andrew James, Gordana Ivosev, Stephen A. Tate, Ron Bonner, Lorne Taylor and Tony Pawson

Abstract: Signalling pathways are commonly organized through inducible protein-protein interactions, mediated by adaptor proteins that link activated receptors to cytoplasmic effectors. However, we have little quantitative data regarding the kinetics with which such networks assemble and dissolve to generate a specific cellular response. We have found that the GRB2 adaptor nucleates a remarkably diverse set of protein complexes, involved in multiple aspects of cellular function. To comprehensively and quantitatively investigate changes in GRB2-based protein interactions in cells, we have designed a mass spectrometry method, AP-SRM (affinity purification-selected reaction monitoring). The data define context-specific and time-dependent networks that form around GRB2 following stimulation, and reveal core and growth factor-selective interaction subsets. These results illustrate the reliability of AP-SRM in the quantitative analysis of dynamic signalling networks; they suggest that capturing a key hub protein and dissecting its interactions by SRM is an approach that can be broadly applied to quantify signalling dynamics.

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