Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Etienne Caron, Romain Roncagalli, Takeshi Hase, Witold E. Wolski, Meena Choi, Marisa G. Menoita, Stephane Durand, Antonio García-Blesa, Ivo Fierro-Monti, Tatjana Sajic, Moritz Heusel, Tobias Weiss, Marie Malissen, Ralph Schlapbach, Ben C. Collins, Samik Ghosh, Hiroaki Kitano, Ruedi Aebersold, Bernard Malissen, Matthias Gstaiger
Spatiotemporal organization of protein interactions in cell signaling is a fundamental process that drives cellular functions. Given differential protein expression across tissues and developmental stages, the architecture and dynamics of signaling interaction proteomes is, likely, highly context dependent. However, current interaction information has been almost exclusively obtained from transformed cells. In this study, we applied an advanced and robust workflow combining mouse genetics and affinity purification (AP)-SWATH mass spectrometry to profile the dynamics of 53 high-confidence protein interactions in primary T cells, using the scaffold protein GRB2 as a model. The workflow also provided a sufficient level of robustness to pinpoint differential interaction dynamics between two similar, but functionally distinct, primary T cell populations. Altogether, we demonstrated that precise and reproducible quantitative measurements of protein interaction dynamics can be achieved in primary cells isolated from mammalian tissues, allowing resolution of the tissue-specific context of cell-signaling events.

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Caron et al. apply an advanced workflow that combines mouse genetic engineering, affinity purification-SWATH mass spectrometry, and open-source software platforms to accurately and reproducibly quantify high-confidence, time-resolved protein interactions in primary T cells isolated from mouse tissues.


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