Global impact of phosphorylation on protein endurance

Abstract

Post-translational modifications such as phosphorylation can have profound effects on the physicochemical and biological properties of proteins. However, high-throughput and systematic approaches have not yet been developed to assess the effects of specific modification types and sites on protein lifetime, which represents a key parameter for understanding signaling rewiring and drug development. Here we describe a proteomic method, DeltaSILAC, to quantify the impact of site-specific phosphorylation on the endurance of thousands of proteins in live cells. Being configured on the reproducible data-independent acquisition mass spectrometry (DIA-MS), the pulse labeling approach using stable isotope-labeled amino acids in cells (SILAC), together with a novel peptide-level matching strategy, this multiplexed assay revealed the global delaying effect of phosphorylation on protein turnover in growing cancer cells. Further, we identified local sequence and structural features in proximity to the phosphorylated sites that could be associated with protein endurance alterations. We found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness and evolutionarily conserved. DeltaSILAC provides a generalizable approach for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology, which is highly complementary to existing methods. Finally, DeltaSILAC is widely applicable to diverse posttranslational modification types and different cell systems.

Protein-Peptide Turnover Profiling reveals wiring of phosphorylation during protein maturation

“…What does it take then, to produce faster clearance of P P compared to P, as has been observed experimentally before (Wu et al, 2020;Zecha et al, 2022)? Arguably, the simplest solution is to relax the assumption that a protein is synthesized in an unmodified form, thus reversing the relative order of the two species (Model 1:2r, Figure 1 G).…”

“…Previous reports measuring turnover of PTM-modified peptides have focused on their potential effects on proteolytic stability, largely equating high measured clearance rates with fast protein degradation (Wu et al, 2020; Zecha et al, 2022), and interpreting PTM sites with fast clearance as potential degrons. In contrast, our theoretical considerations predict that measured changes in clearance can be unrelated to actual protein stability effects.…”

Integrated Proteomic and Metabolomic Analyses of the Mitochondrial Neurodegenerative Disease MELAS

“…Proteomics data were exported in the excel format and further analyzed with R for statistical analysis (t-test) and Spearman's correlation 31 . Metabolomics data from 4 different LC modes were combined for further multivariate statistical analysis.…”