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.

A new paper by the Olsen Group from the Novo Nordisk Foundation Center from Protein Research, describes a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. Due to the use of directDIA and the 60 samples per day method, quantitative data for subcellular proteomes and phospho-proteomes can be generated for six subcellular fractions in just 5 hours of MS time. This enables the possibility to include multiple biological replicates as well as different experimental conditions or time points.




To extend the scope of their spatio-temporal proteomics approach, they employed the methodology to identify subcellular relocation events triggered in response to cellular stress signaling. They treated U2OS cells for one hour with 500 mM sorbitol to induce hyperosmotic stress conditions. Moreover, to study the plasticity of the cells and their recovery from the osmotic stress, they washed out the sorbitol after the hypertonic stress event and collected cells in recovery after 30 minutes, 3 hours and 24 hours, respectively.

Following their high-throughput mapping of the subcellular proteome and phospho-proteome, they were able to quantify 7588 proteins and 9462 phosphorylation-sites. They discovered previously undescribed mechanisms of the cellular stress response using MS-based spatial proteomics. Specifically, that ribotoxicity is impacting ribosome biogenesis and assembly resulting in accumulation of ribosome large subunits (60S) in the nucleolar enriched compartment. Finally, osmotic stress mimics the signalling triggered in vivo by intense muscle contraction. Subcell-analysis of mice muscle after intense exercise also shows translocation of ribosomal subunits between cytosolic and nucleolar compartments.


Collectively, their in vivo and in vitro datasets represent a large resource of subcellular (phospho)-proteome dynamics. Altogether, this manifests the usefulness of the methodology for prospective studies of spatio-temporal regulation using MS-based proteomics.

→ Link: https://www.biorxiv.org/content/10.1101/2021.02.02.425898v1

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