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bioRxiv · 10.1101/2021.02.02.425898

Spatial-proteomics reveal in-vivo phospho-signaling dynamics at subcellular resolution

Martinez del Val, A.·Bekker-Jensen, D. B.·Steigerwald, S.·Mehta, A.·Tran, T.·Sikorski, K.·Torres-Vega, E.·Kwasniewicz, E.·Hlin Brynjolfsdottir, S.·Frankel, L. B.·Kjobsted, R.·Krogh, N.·Lundby, A.·Bekker-Jensen, S.·Lund-Johansen, F.·Olsen, J. V.
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Abstract

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry (MS)-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmarked the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in-vitro in HeLa cells and in-vivo in mouse tissues. Finally, we investigated the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io.

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Martinez del Val, A., Bekker-Jensen, D. B., Steigerwald, S., Mehta, A., Tran, T., Sikorski, K., Torres-Vega, E., Kwasniewicz, E., Hlin Brynjolfsdottir, S., Frankel, L. B., Kjobsted, R., Krogh, N., Lundby, A., Bekker-Jensen, S., Lund-Johansen, F., Olsen, J. V.. 2021-02-02. Spatial-proteomics reveal in-vivo phospho-signaling dynamics at subcellular resolution. https://doi.org/10.1101/2021.02.02.425898

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