title: Selected reaction monitoring mass spectrometry reveals the dynamics of signaling through the GRB2 adaptor.
abstract: Signaling 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 specific cellular responses. To address this deficiency, we designed a mass spectrometry method, affinity purification-selected reaction monitoring (AP-SRM), which we used to comprehensively and quantitatively investigate changes in protein interactions with GRB2, an adaptor protein that participates in a remarkably diverse set of protein complexes involved in multiple aspects of cellular function. Our data reliably define context-specific and time-dependent networks that form around GRB2 after stimulation, and reveal core and growth factor-selective complexes comprising 90 proteins identified as interacting with GRB2 in HEK293T cells. Capturing a key hub protein and dissecting its interactions by SRM should be equally applicable to quantifying signaling dynamics for a range of hubs in protein interaction networks.
authors.affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Joseph and Wolf Lebovic Health Complex, Toronto, Ontario, Canada.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bisson
authors.forename: D Andrew
authors.initials: DA
authors.lastname: James
authors.forename: Gordana
authors.initials: G
authors.lastname: Ivosev
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Tate
authors.forename: Ron
authors.initials: R
authors.lastname: Bonner
authors.forename: Lorne
authors.initials: L
authors.lastname: Taylor
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D051380:GRB2 Adaptor Protein / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human