Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution

• Published in: Nature communications Vol. 12; no. 1; p. 7113
• Main Authors: Martinez-Val, Ana, Bekker-Jensen, Dorte B., Steigerwald, Sophia, Koenig, Claire, Østergaard, Ole, Mehta, Adi, Tran, Trung, Sikorski, Krzysztof, Torres-Vega, Estefanía, Kwasniewicz, Ewa, Brynjólfsdóttir, Sólveig Hlín, Frankel, Lisa B., Kjøbsted, Rasmus, Krogh, Nicolai, Lundby, Alicia, Bekker-Jensen, Simon, Lund-Johansen, Fridtjof, Olsen, Jesper V.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 07.12.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 45; 631/1647/296; 631/337/458/1733; 631/45/475; 631/80/86/2368; 82/58; 96; 96/63; Animal tissues; Animals; Biological Phenomena; Cell Fractionation; Dynamics; Epidermal growth factor receptors; Fractionation; HeLa Cells; Humanities and Social Sciences; Humans; Hypertonicity; In vivo methods and tests; Localization; Male; Mass Spectrometry; Mass spectroscopy; Mice; Mice, Inbred C57BL; multidisciplinary; Muscle contraction; Muscles; Muscular function; Osmotic Pressure; Phosphorylation; Proteins; Proteome - metabolism; Proteomes; Proteomics; Relocation; Ribosomal proteins; Science; Science (multidisciplinary); Signal Transduction; Signaling; Subcellular Fractions - metabolism; Workflow
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-27398-y

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 . Protein activity regulated by phosphorylation can result in subcellular relocation. Here, the authors present a high throughput spatial phosphoproteomics approach to profile six subcellular compartments, providing insights into EGFR and stress signalling dynamics.