Evosep One facilitates rapid and comprehensive maps of the subcellular proteome and phospho-proteome dynamics

Evosep One facilitates rapid and comprehensive maps of the subcellular proteome and phospho-proteome dynamics

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.

Subcellular protein dynamics reveals ribosome accumulation upon osmotic stress

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.

Conclusions 

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

More research on Phosphoproteomics

Here you can see publications available on phosphoproteomics research featuring Evosep One. For a full overview of publications published using the Evosep One Technology visit our Literature room here

TitleSubjectMaterialYearSummaryInstituteEvosep methodMS instrumentationLearn More
High-Throughput Proteomics Quantification Enabled by Fast LC Separation and Advanced PRM Acquisition, , 2018High throughput LC-PRM to monitor the main protein components of AKT/mTOR signaling pathway., ,

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A Compact Quadrupole-Orbitrap Mass Spectrometer With Faims Interface Improves Proteome Coverage In Short Lc Gradients, , , , , , , , , 2020Test of Orbitrap Exploris with DIA and TMT, proteome and phospho., ,

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Fast and reproducible phosphoproteomics using MagReSyn® Amine and Tim-IMAC HP magnetic beads and the Evosep One, , 2020Read this application note to learn about the automated workflows for sample preparation developed by the Olsen group in collaboration with Resyn Biosciences. Protein digestion and phosphoenrichment is fully automated for up to 96 samples in parallel with the Kingfisher Flex robot from Thermo Scientific.

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Rapid proteome analyses using the Evosep One, , , 2018

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High-throughput proteomics with Evosep One, , , , , 2020, ,

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Improving proteome coverage and peptide identification rates in short LC gradients, , , , , 2020, ,

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High throughput proteome and phosphorpoteome sample processing coupled to fast gradient DIA, , , , , , , 2020High throughput workflows for proteomics, ,

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Spatial-proteomics reveal in-vivo phospho-signaling dynamics at subcellular resolution, , , 2021This publication from the Olsen group at the Novo Nordisk Foundation Center for 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.

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Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signatures, , , , 2021In this publication, the Olsen group highlights the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer. They identify protein kinase C delta (PKCδ) inhibition as a strategy to overcome resistance to Notch1 inhibition in T-cell acute lymphoblastic leukemia.,

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