Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution
Ana Martínez‐Val, Dorte B. Bekker‐Jensen, Sophia Steigerwald, Claire Koenig, Ole Østergaard, Adi Mehta, Trung Tran, K. Sikorski, Estefanía Torres-Vega, Ewa Kwasniewicz, Sólveig Hlín Brynjólfsdóttir, Lisa B. Frankel, Rasmus Kjøbsted, Nicolai Krogh, Alicia Lundby, Simon Bekker‐Jensen, Fridtjof Lund‐Johansen, Jesper V. Olsen
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-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in 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 benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate 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 .