Proteome-wide measurements of protein turnover have largely ignored the impact of post-translational modifications (PTMs). To address this gap, we employ stable isotope labelling 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 hypothesize that PTMs with diverging turnover may distinguish states of differential protein stability, structure, localization, enzymatic activity, or protein-protein interactions. We exemplify how the turnover data may facilitate insights into unknown functions of PTMs and provide a web-tool for the scientific community that allows interrogation and visualisation of all turnover data. Since the methodology is applicable to many cell types and modifications, it has substantial potential to prioritize PTMs for functional investigation in the future.

Size exclusion experiments (SEC): Post-translationally modified peptides were targeted in SEC fractions to investigate whether they are enriched in either the monomer or complex fractions of the proteasome. Cycloheximide (CHX) chases: Phosphopeptides were targeted in FBXW7 knock-down or overexpression samples after CHX treatment to validate a potential phosphodegron on TKT. Further, GlyGly (ubiquitin-remnant) peptides of TKT were assayed in CHX chase samples +/- proteasome inhibition to identify ubiquitin-sites that mark TKT for proteasomal degradation. Raw files and msms.txt files used to built spectral libraries can be accessed on the website of the ProteomeXchange Consortium (http://www.proteomexchange.org/) via the PRIDE partner repository with the dataset identifier PXD023218.

SEC: SILAC labelled HeLa or RPMI8226 cells were treated with the proteasome inhibitors MG132 or bortezomib, respectively. Lysates were separated on a SEC column and fractions were subjected to parallel-reaction-monitoring (PRM) measurements on a Thermo HF-X. CHX: FBXW7 was overexpressed or knocked down via siRNA treatment in HEK293 cells. Then, CHX chases were performed and cells were lysed after 0h, 1h, 2h, and 4 h. Additional samples that were treated with MG132 were included for the 2h and 4h treatment durations. For the analysis of phosphodegrons, phosphopeptides were enriched via immobilized metal affinity chromatography. For the measurement of ubiquitinylated peptides, GlyGly was enriched via immunoprecipitation.