Abstract
Interactomes are often measured using affinity purification–mass spectrometry (AP-MS) or yeast two-hybrid approaches, but these methods do not provide stoichiometric or temporal information. We combine quantitative proteomics and size-exclusion chromatography to map 291 coeluting complexes. This method allows mapping of an interactome to the same depth and accuracy as AP-MS with less work and without overexpression or tagging. The use of triplex labeling enables monitoring of interactome rearrangements.
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Acknowledgements
The authors thank members of the Foster group for discussions and advice. This work was supported by a grant from the Canadian Institutes for Health Research to L.J.F. (MOP-77688). L.J.F. is supported by the Canada Research Chairs program and A.R.K. is supported by the Danish Agency for Science Technology and Innovation. Mass spectrometry infrastructure used in this work was supported by the Canada Foundation for Innovation, British Columbia Knowledge Development Fund and BC Proteomics Network.
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A.R.K. conceived of and performed the experiments; A.R.K., J.G. and L.J.F. analyzed the data and wrote the manuscript.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 (PDF 837 kb)
Supplementary Table 1a
Raw chromatograms for proteins identified in the three biological replicates (XLS 2363 kb)
Supplementary Table 2
Limits applied for the three different biological replicates (XLS 19 kb)
Supplementary Table 3
Binary interactions identified (XLS 2762 kb)
Supplementary Table 4
Protein complexes identified (XLS 206 kb)
Supplementary Table 5
Proteins that did change elution time following addition of antibodies against 14-3-3A (XLS 19 kb)
Supplementary Table 6
Proteins identified to have temporal changes after EGF stimulation (XLS 45 kb)
Supplementary Table 7
Proteins that changed elution time following addition of antibody against HRS (XLS 19 kb)
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Kristensen, A., Gsponer, J. & Foster, L. A high-throughput approach for measuring temporal changes in the interactome. Nat Methods 9, 907–909 (2012). https://doi.org/10.1038/nmeth.2131
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DOI: https://doi.org/10.1038/nmeth.2131
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