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Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system

Abstract

Protein complexes and protein interaction networks are essential mediators of most biological functions. Complexes supporting transient functions such as signal transduction processes are frequently subject to dynamic remodeling. Currently, the majority of studies on the composition of protein complexes are carried out by affinity purification and mass spectrometry (AP-MS) and present a static view of the system. For a better understanding of inherently dynamic biological processes, methods to reliably quantify temporal changes of protein interaction networks are essential. Here we used affinity purification combined with sequential window acquisition of all theoretical spectra (AP-SWATH) mass spectrometry to study the dynamics of the 14-3-3β scaffold protein interactome after stimulation of the insulin-PI3K-AKT pathway. The consistent and reproducible quantification of 1,967 proteins across all stimulation time points provided insights into the 14-3-3β interactome and its dynamic changes following IGF1 stimulation. We therefore establish AP-SWATH as a tool to quantify dynamic changes in protein-complex interaction networks.

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Figure 1: AP-SWATH workflow schematic.
Figure 2: Complete quantitative data matrix from AP-SWATH.
Figure 3: 14-3-3β protein interactions are confidently identified by enrichment over control APs.
Figure 4: Quantification of 14-3-3β interactome after perturbation.
Figure 5: AP-SWATH time-resolved data highlight sequestering of the mTORC1 inhibitory subunit AKTS1 but no change in mTORC2 bound to 14-3-3β.
Figure 6: Dynamic range of 14-3-3β–interacting proteins.

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Acknowledgements

We thank P. Navarro for support with data analysis and spectral library generation; C. Ludwig, N. Selevsek and Y. Liu for helpful discussions on SWATH-MS; O. Schubert for the MTB SWATH-MS data file for target assay comparison; S. Hauri and A. van Drogen for advice on cell-line generation, stimulation and APs; A. Kahraman for discussions on structural aspects of 14-3-3; V. Chang for support with SRMstats; and the PRIDE team for assistance with the upload of associated data. We gratefully acknowledge financial support from the SystemsX.ch project PhosphonetX and European Research Council advanced grant Proteomics v3.0 (grant 233226) from the European Union.

Author information

Authors and Affiliations

Authors

Contributions

B.C.C. performed the cell-line generation, cell perturbation, AP and mass spectrometry experiments. L.C.G. co-developed the SWATH-MS methodology and provided critical input on analytical strategy. G.R. and H.L.R. developed the OpenSWATH software and provided critical input on SWATH-MS data analysis strategies. A.V. performed reciprocal AP-MS experiments. M.G. and R.A. conceived of and cosupervised the project. B.C.C. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Ruedi Aebersold.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Notes 1 and 2 and Supplementary Results (PDF 6572 kb)

Supplementary Table 1

Target assays for extraction of SWATH-MS data (XLSX 19778 kb)

Supplementary Table 2

567 high confidence 14-3-3 beta protein interactions (high confidence interactions highlighted in blue, bait in yellow, and control bait in green) (XLSX 264 kb)

Supplementary Table 3

Protein kinases selected for verification by reciprocal AP-MS (XLSX 9 kb)

Supplementary Table 4

Time course dynamics of 567 high confidence 14-3-3 beta protein interactions (XLSX 127 kb)

Supplementary Table 5

Soft clusters from fuzzy c-means clustering of time course (XLSX 24 kb)

Supplementary Table 6

Dynamic range of abundances in 14-3-3 beta protein interactions (bait highlighted in yellow) (XLSX 122 kb)

Supplementary Table 7

Phosphopeptides identified by shotgun-MS analysis of IMAC enrichment from a large pool of 14-3-3 beta affinity purifications from across the time course experiment (XLSX 382 kb)

Supplementary Table 8

Target assays for preliminary extraction of SWATH-MS data from phosphopeptides (XLSX 1699 kb)

Supplementary Table 9

Phosphopeptides identified by AP-SWATH (XLSX 111 kb)

Supplementary Table 10

Phosphosite mapping to 14-3-3 motifs on interacting proteins (XLSX 13 kb)

Supplementary Table 11

Enriched Molecular Function Gene Ontology FAT terms for 567 14-3-3 beta interacting proteins (XLSX 11 kb)

Supplementary Table 12

Enriched InterPro domains for 567 14-3-3 beta interacting proteins (XLSX 12 kb)

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Collins, B., Gillet, L., Rosenberger, G. et al. Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system. Nat Methods 10, 1246–1253 (2013). https://doi.org/10.1038/nmeth.2703

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