To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. 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. Based on the accurate and reproducible mass spectrometry-based method, 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. 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. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.

Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research Shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai 201203 China

Department of Neuro and Sensory Physiology University Medical Center Göttingen 37073 Göttingen Germany

Department of Systems Biology Columbia University New York NY USA

German Cancer Research Center DKFZ 69120 Heidelberg Germany

Yale Cancer Biology Institute Yale University West Haven CT 06516 USA

Yale Cancer Biology Institute Yale University West Haven CT 06516 USA; Department of Genome Integrity Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Yale Cancer Biology Institute Yale University West Haven CT 06516 USA; Department of Pharmacology Yale University School of Medicine New Haven CT 06520 USA

See more in PubMed

Aebersold R, and Mann M (2016). Mass-spectrometric exploration of proteome structure and function. Nature 537, 347–355. PubMed

Bekker-Jensen DB, Bernhardt OM, Hogrebe A, Martinez-Val A, Verbeke L, Gandhi T, Kelstrup CD, Reiter L, and Olsen JV (2020). Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition without the need for spectral libraries. Nature communications 11, 787. PubMed PMC

Betts MJ, Wichmann O, Utz M, Andre T, Petsalaki E, Minguez P, Parca L, Roth FP, Gavin AC, Bork P et al. (2017). Systematic identification of phosphorylation-mediated protein interaction switches. PLoS computational biology 13, e1005462. PubMed PMC

Bruderer R, Bernhardt OM, Gandhi T, Miladinovic SM, Cheng LY, Messner S, Ehrenberger T, Zanotelli V, Butscheid Y, Escher C et al. (2015). Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues. Molecular & cellular proteomics : MCP 14, 1400–1410. PubMed PMC

Bruderer R, Bernhardt OM, Gandhi T, Xuan Y, Sondermann J, Schmidt M, Gomez-Varela D, and Reiter L (2017). Optimization of Experimental Parameters in Data-Independent Mass Spectrometry Significantly Increases Depth and Reproducibility of Results. Molecular & cellular proteomics : MCP 16, 2296–2309. PubMed PMC

Bubb MR, Lenox RH, and Edison AS (1999). Phosphorylation-dependent conformational changes induce a switch in the actin-binding function of MARCKS. J Biol Chem 274, 36472–36478. PubMed

Claydon AJ, and Beynon R (2012). Proteome dynamics: revisiting turnover with a global perspective. Molecular & cellular proteomics : MCP 11, 1551–1565. PubMed PMC

Colaert N, Helsens K, Martens L, Vandekerckhove J, and Gevaert K (2009). Improved visualization of protein consensus sequences by iceLogo. Nature methods 6, 786–787. PubMed

Collins BC, Hunter CL, Liu Y, Schilling B, Rosenberger G, Bader SL, Chan DW, Gibson BW, Gingras AC, Held JM, et al. (2017). Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry. Nature communications 8, 291. PubMed PMC

Cox J, and Mann M (2008). MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature biotechnology 26, 1367–1372. PubMed

Fornasiero EF, Mandad S, Wildhagen H, Alevra M, Rammner B, Keihani S, Opazo F, Urban I, Ischebeck T, Sakib MS, et al. (2018). Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions. Nature communications 9, 4230. PubMed PMC

Gao Q, Zhu H, Dong L, Shi W, Chen R, Song Z, Huang C, Li J, Dong X, Zhou Y et al. (2019). Integrated Proteogenomic Characterization of HBV-Related Hepatocellular Carcinoma. Cell 179, 561–577 e522. PubMed

Gillet LC, Navarro P, Tate S, Rost H, Selevsek N, Reiter L, Bonner R, and Aebersold R (2012). Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Molecular & cellular proteomics : MCP 11, O111 016717. PubMed PMC

Gotzke H, Kilisch M, Martinez-Carranza M, Sograte-Idrissi S, Rajavel A, Schlichthaerle T, Engels N, Jungmann R, Stenmark P, Opazo F et al. (2019). The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nature communications 10, 4403. PubMed PMC

Hein JB, Hertz EPT, Garvanska DH, Kruse T, and Nilsson J (2017). Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis. Nat Cell Biol 19, 1433–1440. PubMed

Hidalgo San Jose L, and Signer RAJ (2019). Cell-type-specific quantification of protein synthesis in vivo. Nature protocols 14, 441–460. PubMed PMC

Holt LJ (2012). Regulatory modules: Coupling protein stability to phopshoregulation during cell division. FEBS Lett 586, 2773–2777. PubMed

Huang da W, Sherman BT, and Lempicki RA (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature protocols 4, 44–57. PubMed

Huang JX, Lee G, Cavanaugh KE, Chang JW, Gardel ML, and Moellering RE (2019). High throughput discovery of functional protein modifications by Hotspot Thermal Profiling. Nature methods 16, 894–901. PubMed PMC

Hunter T (2007). The age of crosstalk: phosphorylation, ubiquitination, and beyond. Molecular cell 28, 730–738. PubMed

Jovanovic M, Rooney MS, Mertins P, Przybylski D, Chevrier N, Satija R, Rodriguez EH, Fields AP, Schwartz S, Raychowdhury R et al. (2015). Immunogenetics. Dynamic profiling of the protein life cycle in response to pathogens. Science 347, 1259038. PubMed PMC

Kholodenko BN (2006). Cell-signalling dynamics in time and space. Nat Rev Mol Cell Biol 7, 165–176. PubMed PMC

Kiyatkin A, van Alderwerelt van Rosenburgh IK, Klein DE, and Lemmon MA (2020). Kinetics of receptor tyrosine kinase activation define ERK signaling dynamics. Sci Signal 13. PubMed PMC

Krahmer N, Najafi B, Schueder F, Quagliarini F, Steger M, Seitz S, Kasper R, Salinas F, Cox J, Uhlenhaut NH, et al. (2018). Organellar Proteomics and Phospho-Proteomics Reveal Subcellular Reorganization in Diet-Induced Hepatic Steatosis. Developmental cell 47, 205–221 e207. PubMed

Krug K, Mertins P, Zhang B, Hornbeck P, Raju R, Ahmad R, Szucs M, Mundt F, Forestier D, Jane-Valbuena J, et al. (2019). A Curated Resource for Phosphosite-specific Signature Analysis. Molecular & cellular proteomics : MCP 18, 576–593. PubMed PMC

Kruger M, Moser M, Ussar S, Thievessen I, Luber CA, Forner F, Schmidt S, Zanivan S, Fassler R, and Mann M (2008). SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function. Cell 134, 353–364. PubMed

Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, and Marra MA (2009). Circos: an information aesthetic for comparative genomics. Genome research 19, 1639–1645. PubMed PMC

Lahiry P, Torkamani A, Schork NJ, and Hegele RA (2010). Kinase mutations in human disease: interpreting genotype-phenotype relationships. Nature reviews Genetics 11, 60–74. PubMed

Li W, Chi H, Salovska B, Wu C, Sun L, Rosenberger G, and Liu Y (2019). Assessing the Relationship Between Mass Window Width and Retention Time Scheduling on Protein Coverage for Data-Independent Acquisition. J Am Soc Mass Spectrom 30, 1396–1405. PubMed

Lim MY, Paulo JA, and Gygi SP (2019). Evaluating False Transfer Rates from the Match-between-Runs Algorithm with a Two-Proteome Model. Journal of proteome research 18, 4020–4026. PubMed PMC

Liu Y, Beyer A, and Aebersold R (2016). On the Dependency of Cellular Protein Levels on mRNA Abundance. Cell 165, 535–550. PubMed

Liu Y, Borel C, Li L, Muller T, Williams EG, Germain PL, Buljan M, Sajic T, Boersema PJ, Shao W, et al. (2017). Systematic proteome and proteostasis profiling in human Trisomy 21 fibroblast cells. Nature communications 8, 1212. PubMed PMC

Liu Y, Mi Y, Mueller T, Kreibich S, Williams EG, Van Drogen A, Borel C, Frank M, Germain PL, Bludau I, et al. (2019). Multi-omic measurements of heterogeneity in HeLa cells across laboratories. Nature biotechnology 37, 314–322. PubMed

Mandad S, Rahman RU, Centeno TP, Vidal RO, Wildhagen H, Rammner B, Keihani S, Opazo F, Urban I, Ischebeck T, et al. (2018). The codon sequences predict protein lifetimes and other parameters of the protein life cycle in the mouse brain. Sci Rep 8, 16913. PubMed PMC

Mann M, and Jensen ON (2003). Proteomic analysis of post-translational modifications. Nature biotechnology 21, 255–261. PubMed

Martin-Perez M, and Villen J (2017). Determinants and Regulation of Protein Turnover in Yeast. Cell Syst 5, 283–294 e285. PubMed PMC

McShane E, Sin C, Zauber H, Wells JN, Donnelly N, Wang X, Hou J, Chen W, Storchova Z, Marsh JA, et al. (2016). Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation. Cell 167, 803–815 e821. PubMed

Mehnert M, Li W, Wu C, Salovska B, and Liu Y (2019). Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1. Proteomics, e1800438. PubMed

Metz KS, Deoudes EM, Berginski ME, Jimenez-Ruiz I, Aksoy BA, Hammerbacher J, Gomez SM, and Phanstiel DH (2018). Coral: Clear and Customizable Visualization of Human Kinome Data. Cell systems 7, 347–350 e341. PubMed PMC

Meyer JG, Mukkamalla S, Steen H, Nesvizhskii AI, Gibson BW, and Schilling B (2017). PIQED: automated identification and quantification of protein modifications from DIA-MS data. Nature methods 14, 646–647. PubMed PMC

Ng PC, and Henikoff S (2003). SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 31, 3812–3814. PubMed PMC

Nguyen LK, Kolch W, and Kholodenko BN (2013). When ubiquitination meets phosphorylation: a systems biology perspective of EGFR/MAPK signalling. Cell Commun Signal 11, 52. PubMed PMC

Ochoa D, Jarnuczak AF, Vieitez C, Gehre M, Soucheray M, Mateus A, Kleefeldt AA, Hill A, Garcia-Alonso L, Stein F et al. (2020). The functional landscape of the human phosphoproteome. Nature biotechnology 38, 365–373. PubMed PMC

Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, and Mann M (2006). Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648. PubMed

Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, and Mann M (2002). Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Molecular & cellular proteomics : MCP 1, 376–386. PubMed

Pratt JM, Petty J, Riba-Garcia I, Robertson DH, Gaskell SJ, Oliver SG, and Beynon RJ (2002). Dynamics of protein turnover, a missing dimension in proteomics. Molecular & cellular proteomics : MCP 1, 579–591. PubMed

Riman S, Rizkallah R, Kassardjian A, Alexander KE, Luscher B, and Hurt MM (2012). Phosphorylation of the transcription factor YY1 by CK2alpha prevents cleavage by caspase 7 during apoptosis. Mol Cell Biol 32, 797–807. PubMed PMC

Rosenberger G, Bludau I, Schmitt U, Heusel M, Hunter CL, Liu Y, MacCoss MJ, MacLean BX, Nesvizhskii AI, Pedrioli PGA, et al. (2017a). Statistical control of peptide and protein error rates in large-scale targeted data-independent acquisition analyses. Nature methods 14, 921–927. PubMed PMC

Rosenberger G, Liu Y, Rost HL, Ludwig C, Buil A, Bensimon A, Soste M, Spector TD, Dermitzakis ET, Collins BC, et al. (2017b). Inference and quantification of peptidoforms in large sample cohorts by SWATH-MS. Nature biotechnology 35, 781–788. PubMed PMC

Rost HL, Liu Y, D’Agostino G, Zanella M, Navarro P, Rosenberger G, Collins BC, Gillet L, Testa G, Malmstrom L, et al. (2016). TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics. Nature methods 13, 777–783. PubMed PMC

Salovska B, Zhu H, Gandhi T, Frank M, Li W, Rosenberger G, Wu C, Germain PL, Zhou H, Hodny Z, et al. (2020). Isoform-resolved correlation analysis between mRNA abundance regulation and protein level degradation. Molecular systems biology 16, e9170. PubMed PMC

Schluter C, Duchrow M, Wohlenberg C, Becker MH, Key G, Flad HD, and Gerdes J (1993). The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J Cell Biol 123, 513–522. PubMed PMC

Schneider CA, Rasband WS, and Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nature methods 9, 671–675. PubMed PMC

Schwanhausser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, and Selbach M (2011). Global quantification of mammalian gene expression control. Nature 473, 337–342. PubMed

Searle BC, Lawrence RT, MacCoss MJ, and Villen J (2019). Thesaurus: quantifying phosphopeptide positional isomers. Nature methods 16, 703–706. PubMed PMC

Shaner NC, Lambert GG, Chammas A, Ni Y, Cranfill PJ, Baird MA, Sell BR, Allen JR, Day RN, Israelsson M et al. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nature methods 10, 407–409. PubMed PMC

Skowyra D, Craig KL, Tyers M, Elledge SJ, and Harper JW (1997). F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91, 209–219. PubMed

Smith LM, and Kelleher NL (2018). Proteoforms as the next proteomics currency. Science 359, 1106–1107. PubMed PMC

Smith LM, Kelleher NL, and Consortium for Top Down, P. (2013). Proteoform: a single term describing protein complexity. Nature methods 10, 186–187. PubMed PMC

Suizu F, Hiramuki Y, Okumura F, Matsuda M, Okumura AJ, Hirata N, Narita M, Kohno T, Yokota J, Bohgaki M et al. (2009). The E3 ligase TTC3 facilitates ubiquitination and degradation of phosphorylated Akt. Developmental cell 17, 800–810. PubMed

Swaney DL, Beltrao P, Starita L, Guo A, Rush J, Fields S, Krogan NJ, and Villen J (2013). Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation. Nature methods 10, 676–682. PubMed PMC

Szymczak-Workman AL, Vignali KM, and Vignali DA (2012). Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb Protoc 2012, 199–204. PubMed

Thul PJ, Akesson L, Wiking M, Mahdessian D, Geladaki A, Ait Blal H, Alm T, Asplund A, Bjork L, Breckels LM, et al. (2017). A subcellular map of the human proteome. Science 356. PubMed

Toyama BH, Savas JN, Park SK, Harris MS, Ingolia NT, Yates JR 3rd, and Hetzer MW (2013). Identification of long-lived proteins reveals exceptional stability of essential cellular structures. Cell 154, 971–982. PubMed PMC

van der Wal L, Bezstarosti K, Sap KA, Dekkers DHW, Rijkers E, Mientjes E, Elgersma Y, and Demmers JAA (2018). Improvement of ubiquitylation site detection by Orbitrap mass spectrometry. Journal of proteomics 172, 49–56. PubMed

Vaser R, Adusumalli S, Leng SN, Sikic M, and Ng PC (2016). SIFT missense predictions for genomes. Nature protocols 11, 1–9. PubMed

Venable JD, Dong MQ, Wohlschlegel J, Dillin A, and Yates JR (2004). Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra. Nature methods 1, 39–45. PubMed

Verma R, Annan RS, Huddleston MJ, Carr SA, Reynard G, and Deshaies RJ (1997). Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase. Science 278, 455–460. PubMed

Verweij FJ, Bebelman MP, Jimenez CR, Garcia-Vallejo JJ, Janssen H, Neefjes J, Knol JC, de Goeij-de Haas R, Piersma SR, Baglio SR et al. (2018). Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling. J Cell Biol 217, 1129–1142. PubMed PMC

Wang S, Cai Y, Cheng J, Li W, Liu Y, and Yang H (2019). motifeR: An Integrated Web Software for Identification and Visualization of Protein Posttranslational Modification Motifs. Proteomics 19, e1900245. PubMed

Wang S, Li W, Liu S, and Xu J (2016). RaptorX-Property: a web server for protein structure property prediction. Nucleic acids research 44, W430–435. PubMed PMC

Welle KA, Zhang T, Hryhorenko JR, Shen S, Qu J, and Ghaemmaghami S (2016). Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing. Molecular & cellular proteomics : MCP 15, 3551–3563. PubMed PMC

Xu JY, Zhang C, Wang X, Zhai L, Ma Y, Mao Y, Qian K, Sun C, Liu Z, Jiang S et al. (2020). Integrative Proteomic Characterization of Human Lung Adenocarcinoma. Cell 182, 245–261 e217. PubMed

Zecha J, Meng C, Zolg DP, Samaras P, Wilhelm M, and Kuster B (2018). Peptide Level Turnover Measurements Enable the Study of Proteoform Dynamics. Molecular & cellular proteomics : MCP 17, 974–992. PubMed PMC