Abstract
Proteome-wide measurements of protein turnover have largely ignored the impact of post-translational modifications (PTMs). To address this gap, we employ stable isotope labeling and mass spectrometry to measure the turnover of >120,000 peptidoforms including >33,000 phosphorylated, acetylated, and ubiquitinated peptides for >9,000 native proteins. This site-resolved protein turnover (SPOT) profiling discloses global and site-specific differences in turnover associated with the presence or absence of PTMs. While causal relationships may not always be immediately apparent, we speculate that PTMs with diverging turnover may distinguish states of differential protein stability, structure, localization, enzymatic activity, or protein-protein interactions. We show examples of how the turnover data may give insights into unknown functions of PTMs and provide a freely accessible online tool that allows interrogation and visualisation of all turnover data. The SPOT methodology is applicable to many cell types and modifications, offering the potential to prioritize PTMs for future functional investigations.
Highlights
Proteome-wide measurements of protein turnover have largely ignored the impact of posttranslational modifications (PTMs)
HeLa cells were pulsed with SILAC medium in quadruplicates including two label swap experiments: For two replicates, medium containing light lysine (K0) and arginine (R0) on unlabeled cells was exchanged with medium containing 13C and 15N labeled, heavy lysine (K10) and arginine (R10; exemplified in Fig. 1a, b)
Phosphorylated, di-glycinemodified and acetylated peptides were sequentially enriched followed by the quantification of light and heavy labeled peptides by mass spectrometry (Fig. 1a)
Summary
Proteome-wide measurements of protein turnover have largely ignored the impact of posttranslational modifications (PTMs) To address this gap, we employ stable isotope labeling and mass spectrometry to measure the turnover of >120,000 peptidoforms including >33,000 phosphorylated, acetylated, and ubiquitinated peptides for >9,000 native proteins. We employ stable isotope labeling and mass spectrometry to measure the turnover of >120,000 peptidoforms including >33,000 phosphorylated, acetylated, and ubiquitinated peptides for >9,000 native proteins This site-resolved protein turnover (SPOT) profiling discloses global and site-specific differences in turnover associated with the presence or absence of PTMs. While causal relationships may not always be immediately apparent, we speculate that PTMs with diverging turnover may distinguish states of differential protein stability, structure, localization, enzymatic activity, or protein-protein interactions. Phosphorylation, acetylation, and ubiquitination SPOT profiling suggests that modification sites that differ in turnover from the underlying bulk protein are involved in diverse cellular processes that go beyond immediate protein stabilization or destabilization. To illustrate the utility of our data as a resource and to highlight PTMs of potential regulatory relevance, we discuss examples of sites that may be involved in protein interactions, localization, enzymatic activity or degradation
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
