Spatial-proteomics revealin-vivophospho-signaling dynamics at subcellular resolution

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 (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. 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 benchmarked the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in-vitro in HeLa cells and in-vivo in mouse tissues. Finally, we investigated 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.

Deep and fast label-free Dynamic Organellar Mapping

“…Based on our extensive optimization of LC-MS formats, we now recommend these two formats for organellar maps: 1) Deep measurement for biological comparisons with 12 hours MS time per map, either by single shot injections on a 100 min nanoLC gradient or triple shot injections on a 44 min Evosep gradient; 2) High throughput maps for pilots and method optimization with 2.5 hours per map on Evosep 21 min gradients. The latter format has recently been used by the Olsen-lab [8] for their chemical fractionation-based spatial proteomics method and achieved a similar depth to our study, suggesting that this is a robust LC-MS approach with consistent depth across machines and even sites.…”

“…Therefore, protein localization must be tightly regulated to ensure correct protein function and, conversely, a large variety of human diseases have been linked to disrupted protein transport (reviewed in [1,2]). Our understanding of protein function is thus incomplete without a precise view of the dynamics of protein movement within the cell, fueling interest in the study of the spatial proteome [3][4][5][6][7][8][9].…”

“…Moreover, the MS2 fragment ions can be used for quantification in addition to the MS1 peptide intensities, increasing the precision of quantification [24]. For these reasons, DIA is becoming the strategy of choice for extensive profiling-based approaches such as SEC-MS [25] and has recently been applied to high-throughput subcellular phosphoproteomics [8].…”

Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution

“…Custom R code used in the manuscript is available in the GitHub at https://github.com/SpatialProteoDynamics/SpatialProteoDynamics.github.io and via Zenodo 65 at 10.5281/zenodo.5635633. PTM collapse plugin requires Perseus and R (minimum version 3.6.0) to run and it is available at https://github.com/AlexHgO/Perseus_Plugin_Peptide_Collapse .…”

Subcellular Transcriptomics and Proteomics: A Comparative Methods Review

“…The technique was expanded for global organelle analyses in multiple mouse tissues 246,247 . Another density gradient centrifugation technique, Localization of Organelle Proteins by Isotope Tagging (LOPIT), employed isotope-coded affinity tagging (ICAT) to multiplex the gradient fractions and map the global subcellular proteome of the Arabidopsis thaliana root-derived callus material 248,249 . Since then, LOPIT has evolved alongside isobaric tagging technologies, allowing the study of the subcellular proteomes of diverse model systems, including human cell lines, chicken lymphocytes, and D. melanogaster embryos [250][251][252][253] .…”