Spatial-proteomics reveals phospho-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-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.

Rapid and in-depth coverage of the (phospho-)proteome with deep libraries and optimal window design for dia-PASEF

“…Most are known to be critical for intact EGF signaling. For example, we detected phosphorylation of T693, Y1110, Y1172, Y1197 on the receptor EGFR itself, Y427 on the adaptor protein Src Homology 2 Domain-Containing-Transforming Protein C1 (SHC1), Y659 on Growth Factor Receptor Bound Protein 2-Associated Protein 1 (GAB1) and on the downstream kinases Mitogen-Activated Protein Kinase 2 (MAP2K2) (T394) and Mitogen-Activated Protein Kinase 1 and 3 (MAPK1/3) (T185/Y187, T202/Y204) (53) (Fig. 6C, E).…”

Deep and fast label-free Dynamic Organellar Mapping

“…Our extensive protocols [42], in conjunction with our DOM-QC tool, further facilitate rapid method establishment. Of note, the Olsen lab recently introduced a fast spatial proteomics method based on chemical fractionation [9], which provides complementary insights to our centrifugation-based profiling of intact organelles. Their approach also utilizes 21 min Evosep gradients, as implemented here for rapid DIA-DOMs, and achieved a similar depth to our method, suggesting that this is a robust LC-MS approach with consistent performance across sites.…”

Proximity-Dependent Biotinylation Approaches to Explore the Dynamic Compartmentalized Proteome

“…Comparatively fewer studies have globally analyzed PTM distributions across organelles to reveal PTM-specific localizations and/or functions, though some studies have begun to shed light on this aspect of regulation (Zhou et al, 2010;Hebert et al, 2013;Still et al, 2013;Christoforou et al, 2016;Krahmer et al, 2018;Lundberg and Borner 2019). For example, a recent study that combined sequential cell fractionation (resolving six subcellular fractions) with phosphopeptide enrichment in cells and mice revealed the rapid relocalization of ribosomal subunits to the nucleolus in response to hypertonicity and muscle contraction, and alterations in the phosphorylation of ribosome assembly factors (Martinez-Val et al, 2021). Specific phosphorylated forms of ribosomal subunits (e.g., ribosomal protein S6 (RPS6) triphosphorylated at S235/S236/S240) were also found in different cell fractions than their nonphosphorylated counterparts (Martinez-Val et al, 2021), suggesting that this strategy can reveal correlations between a protein's localization and its PTMs, potentially paving the way for mechanistic studies.…”

“…For example, a recent study that combined sequential cell fractionation (resolving six subcellular fractions) with phosphopeptide enrichment in cells and mice revealed the rapid relocalization of ribosomal subunits to the nucleolus in response to hypertonicity and muscle contraction, and alterations in the phosphorylation of ribosome assembly factors (Martinez-Val et al, 2021). Specific phosphorylated forms of ribosomal subunits (e.g., ribosomal protein S6 (RPS6) triphosphorylated at S235/S236/S240) were also found in different cell fractions than their nonphosphorylated counterparts (Martinez-Val et al, 2021), suggesting that this strategy can reveal correlations between a protein's localization and its PTMs, potentially paving the way for mechanistic studies. These studies are however challenging: to simultaneously obtain the organellar separation and the depth of proteome coverage requires access to significant instrument time, and for all procedures to be optimized within a laboratory.…”