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A high-throughput approach for measuring temporal changes in the interactome

Nature Methods volume 9, pages 907–909 (2012)Cite this article

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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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Figure 1: Identification of spatiotemporal changes in the interactome after EGF stimulation.
Figure 2: Evaluation of the PCP-SILAC approach to identify protein-protein interactions.
Figure 3: Determination of stoichiometry and the interactome response to EGF stimulation.

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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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Authors and Affiliations

  1. Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada

    Anders R Kristensen, Joerg Gsponer & Leonard J Foster

  2. Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia, Canada

    Anders R Kristensen, Joerg Gsponer & Leonard J Foster

Authors
  1. Anders R Kristensen
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  2. Joerg Gsponer
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  3. Leonard J Foster
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Contributions

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.

Corresponding author

Correspondence to Leonard J Foster.

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Competing interests

The authors declare no competing financial interests.

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 1b (XLS 2660 kb)

Supplementary Table 1c (XLS 6851 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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