Abstract
Signaling pathways are commonly organized through inducible protein-protein interactions, mediated by adaptor proteins that link activated receptors to cytoplasmic effectors1. 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.
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Acknowledgements
The authors would like to thank K. Colwill and A.-C. Gingras for insightful comments on the manuscript, R. Bagshaw, G. Findlay, J. Gish, J. Jin, B. Larsen and O. Rocks for discussions and A. Yu Dai, B. Larsen and B. Liu for technical assistance. This project is supported through Ontario Research Fund grants from the Ontario Ministry of Research and Innovation and grants from Genome Canada through the Ontario Genomics Institute, the Canadian Institutes of Health Research (CIHR, grant MOP-6849), the Terry Fox Research Institute/Ontario Institute for Cancer Research and the Canadian Cancer Society Research Institute. N.B. holds a research fellowship from the CIHR. T.P. holds the Apotex Chair in Molecular Oncology and is a Distinguished Investigator of the CIHR.
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G.I., S.A.T. and R.B. are employees of AB Sciex. AB Sciex has provided support for Ontario Research Fund grants (awarded to T.P.).
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Supplementary Text and Figures
Supplementary Tables 1–5, Supplementary Methods and Supplementary Figsures 1–20 (PDF 2350 kb)
Supplementary Data 1
Statistical analysis at the protein level of all AP-SRM experiments. (XLS 326 kb)
Supplementary Data 2
Statiscal analysis at the peptide level of all AP-SRM experiments. (XLS 606 kb)
Supplementary Data 3
Experimental evidence for phosphorylation sites data presented in Supplementary Table 2. (XLS 227 kb)
Supplementary Data 4
List of all proteins identified in GRB2 and control GFP AP-MS experiments. (XLS 149 kb)
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Bisson, N., James, D., Ivosev, G. et al. Selected reaction monitoring mass spectrometry reveals the dynamics of signaling through the GRB2 adaptor. Nat Biotechnol 29, 653–658 (2011). https://doi.org/10.1038/nbt.1905
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DOI: https://doi.org/10.1038/nbt.1905
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