![]() ![]() Yet, acetylation alone can also result in the degradation of other transcriptional regulators, such as HIF1α and GATA-1 16, 17 which illustrates the diversity and complexity of the underlying regulatory mechanisms. In contrast to such positive PTM crosstalk, a competitive interplay between stabilizing acetylation and destabilizing ubiquitination has been suggested for several transcription factors including p53 and MYC 13– 15. ![]() Such phosphodegrons are, for instance, responsible for the periodic degradation of cell cycle regulators to enable the progression of cell proliferation 11, 12. Phosphorylated motifs that serve as recognition elements for certain E3 ubiquitin ligases can mediate ubiquitination and subsequent protein degradation. Most prominently, polyubiquitin chains linked via the side chain of lysine at position 48 typically mark proteins for proteasomal degradation 10. For some proteins, post-translational modifications (PTMs) have been implicated in regulating protein stability. This demonstrates the high degree of regulation exerted on protein synthesis and degradation processes. Protein half-lives computed from such data have been found to range from minutes to weeks and vary considerably between cell types and tissues 7– 9. Turnover of cellular proteins is determined by tracing the incorporation and loss of isotopically labeled amino acids into proteins in a time-resolved manner. More recently, so-called dynamic or pulsed SILAC (stable isotope labeling by amino acids in cell culture) approaches together with mass spectrometry-based readouts have enabled the investigation of endogenous protein turnover on a proteome-wide scale 6. The SPOT methodology is applicable to many cell types and modifications, offering the potential to prioritize PTMs for future functional investigations.Įver since the continuous turnover of body constituents was discovered eight decades ago 1, the dynamic nature of cellular proteins has been studied extensively 2, and its significance to maintain protein homeostasis has been widely acknowledged 3– 5. 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. 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. This site-resolved protein turnover (SPOT) profiling discloses global and site-specific differences in turnover associated with the presence or absence of 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. Proteome-wide measurements of protein turnover have largely ignored the impact of post-translational modifications (PTMs). ![]() Source data are provided with this paper. Previously published potein crystal structures were obtained from the RCSB protein databank website (PDB IDs: 5LE5, 3B97, 3MOS ). Source data of all figures are provided with this paper. In addition, all analysed data of modified and counterpart sites can be viewed and explored via .de. Normalized and further processed data such as turnover rates and N / P ratios and nucleotide sequences are provided in the Supplementary Data files. Raw data and results of the PRM assays can also be examined on the Panorama Public website at (SEC and CHX experiments). The MS proteomics raw data and MaxQuant search results including the fasta file from the Swiss-Prot database have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository under the accession codes PXD023218 (dSILAC, dSILAC-TMT, and SEC experiments) and PXD023234 (SEC and CHX PRM assays). ![]()
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