In the last decade, mass spectrometry (MS) based proteomics has greatly facilitated the analysis of proteins and their PTMs ( Aebersold and Mann, 2016 Mann and Jensen, 2003). However, high-throughput discovery tools are currently lacking to illustrate how phosphorylated proteins are degraded by proteostasis and proteolysis pathways. Specifically, the functional crosstalk between phosphorylation and ubiquitination was discovered to substantially downregulate the levels of key phosphoproteins in a variety of pathways such as EGFR/MAPK signaling ( Nguyen et al., 2013 Suizu et al., 2009) and cell-cycle control ( Skowyra et al., 1997 Verma et al., 1997). Such a “phosphate-transfer independent” mechanism was shown to enable the rewiring of cellular biochemical states after adaptation ( Hunter, 2007 Nguyen et al., 2013) and the establishment of cell fitness against intrinsic genetic alterations ( Lahiry et al., 2010 Ochoa et al., 2020). We aim to focus on this important regulatory mechanism in the present study. Nevertheless, the long-term regulation of turnover and decay of those constitutively phosphorylated proteins is also critical for the cellular systems. In order to adapt to temporal environmental changes, cells have to utilize kinases and phosphatases to effectively and instantly catalyze phosphate-transfer between substrates. However, the impact of phosphorylation sites on protein turnover has so far not been assessed at the proteome-scale. Phosphorylation is a critical PTM, which has been demonstrated to be essential for signaling transduction ( Olsen et al., 2006), mediating protein-protein interaction ( Betts et al., 2017), and altering the three-dimensional protein structure, thermal stability ( Huang et al., 2019), and subcellular localization ( Krahmer et al., 2018) – frequently in a modification site-specific manner. Protein post-translational modifications (PTMs) can alter the above properties, leading to diverse functions ( Smith and Kelleher, 2018 Smith et al., 2013). As the primary functional biomolecule involved in most cellular processes, proteins have been characterized by various properties, such as structure, abundance, localization, stability, and turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein expression lifetime in the context of cell signaling and disease biology.īiological signaling networks receive and transduce signals based on both amplitude and duration ( Kholodenko, 2006). We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved whereas the Glutamic acids surrounding phosphosites significantly delay protein turnover. Based on the accurate and reproducible mass spectrometry, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. ![]() To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated.
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