Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution

• DOI: 10.1038/s41467-021-27398-y
• Publication Date: 2021-12-07T11:02:38Z
• Authors: Ana Martinez-Val; Dorte B. Bekker-J...; Sophia Steigerwald; Claire Koenig; Ole Østergaard; Adi Mehta; Trung Tran; Krzysztof Sikorski; Estefanía Torres-...; Ewa Kwasniewicz; Sólveig Hlín Bryn...; Lisa B. Frankel; Rasmus Kjøbsted; Nicolai Krogh; Alicia Lundby; Simon Bekker-Jensen; Fridtjof Lund-Joh...; Jesper V. Olsen
• Keywords: Male; Proteomics; 0301 basic medicine; Proteome; Science; Q; Cell Fractionation; Article; Mass Spectrometry; Workflow; Mice, Inbred C57BL; Mice; 03 medical and health sciences; Osmotic Pressure; Animals; Humans; Phosphorylation; Biological Phenomena; HeLa Cells; Signal Transduction; Subcellular Fractions

AbstractDynamic 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.