Skip to main content
SpringerLink
Log in

Mass Spectrometry-Based Proteomics for Quantifying DNA Damage-Induced Phosphorylation

  • Protocol
  • First Online:
ATM Kinase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1599))

Abstract

Protein phosphorylation plays central regulatory roles in DNA damage repair and signaling. Protein kinases of the phosphatidylinositol 3-kinase-related kinase family ATM, ATR, and DNA-PKcs mediate phosphorylation of hundreds of substrates after DNA damage and thereby orchestrate the cellular response to DNA damage. Protein phosphorylation can be studied using antibodies that specifically recognize phosphorylated protein species; however, this approach is limited by existing antibodies and does not permit unbiased discovery of phosphorylation sites or analyzing phosphorylation sites in a high-throughput manner. Mass spectrometry (MS)-based proteomics has emerged as a powerful method for identification of phosphorylation sites on individual proteins and proteome-wide. To identify phosphorylation sites, proteins are digested into peptides and phosphopeptides are enriched using titanium dioxide (TiO2)-based chromatography followed by the identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantitative proteomics approaches, such as stable isotope labeling with amino acids in cell culture (SILAC), enable relative quantification of phosphopeptide abundance in different conditions. Here, we describe a streamlined protocol for enrichment of phosphopeptides using TiO2-based chromatography, and outline the application of quantitative phosphoproteomics for the identification of DNA damage-induced phosphorylation and substrates of kinases functioning after DNA damage.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Polo SE, Jackson SP (2011) Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev 25:409–433. doi:10.1101/gad.2021311

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40:179–204. doi:10.1016/j.molcel.2010.09.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Beli P, Lukashchuk N, Wagner SA et al (2012) Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response. Mol Cell 46:212–225. doi:10.1016/j.molcel.2012.01.026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Matsuoka S, Ballif BA, Smogorzewska A et al (2007) ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316:1160–1166. doi:10.1126/science.1140321

    Article  CAS  PubMed  Google Scholar 

  5. Bennetzen MV, Larsen DH, Bunkenborg J et al (2010) Site-specific phosphorylation dynamics of the nuclear proteome during the DNA damage response. Mol Cell Proteomics 9:1314–1323. doi:10.1074/mcp.M900616-MCP200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bensimon A, Schmidt A, Ziv Y et al (2010) ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage. Sci Signal 3:rs3. doi:10.1126/scisignal.2001034

    Article  CAS  PubMed  Google Scholar 

  7. Stokes MP, Rush J, MacNeill J et al (2007) Profiling of UV-induced ATM/ATR signaling pathways. Proc Natl Acad Sci 104:19855–19860. doi:10.1073/pnas.0707579104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Blasius M, Forment JV, Thakkar N et al (2011) A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1. Genome Biol 12:R78. doi:10.1186/gb-2011-12-8-r78

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wagner SA, Oehler H, Voigt A et al (2015) ATR inhibition rewires cellular signaling networks induced by replication stress. Proteomics. doi:10.1002/pmic.201500172

    Google Scholar 

  10. Bensimon A, Aebersold R, Shiloh Y (2011) Beyond ATM: the protein kinase landscape of the DNA damage response. FEBS Lett 585:1625–1639. doi:10.1016/j.febslet.2011.05.013

    Article  CAS  PubMed  Google Scholar 

  11. Larance M, Lamond AI (2015) Multidimensional proteomics for cell biology. Nat Rev Mol Cell Biol 16:269–280. doi:10.1038/nrm3970

    Article  CAS  PubMed  Google Scholar 

  12. Macek B, Mann M, Olsen JV (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49:199–221. doi:10.1146/annurev.pharmtox.011008.145606

    Article  CAS  PubMed  Google Scholar 

  13. Thingholm TE, Jensen ON, Larsen MR (2009) Analytical strategies for phosphoproteomics. Proteomics 9:1451–1468. doi:10.1002/pmic.200800454

    Article  CAS  PubMed  Google Scholar 

  14. Nita-Lazar A, Saito-Benz H, White FM (2008) Quantitative phosphoproteomics by mass spectrometry: past, present, and future. Proteomics 8:4433–4443. doi:10.1002/pmic.200800231

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rigbolt KTG, Blagoev B (2012) Quantitative phosphoproteomics to characterize signaling networks. Semin Cell Dev Biol 23:863–871. doi:10.1016/j.semcdb.2012.05.006

    Article  CAS  PubMed  Google Scholar 

  16. Engholm-Keller K, Larsen MR (2011) Titanium dioxide as chemo-affinity chromatographic sorbent of biomolecular compounds—applications in acidic modification-specific proteomics. J Proteomics 75:317–328. doi:10.1016/j.jprot.2011.07.024

    Article  CAS  PubMed  Google Scholar 

  17. Zhou H, Low TY, Hennrich ML et al (2011) Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment. Mol Cell Proteomics 10:M110.006452. doi:10.1074/mcp.M110.006452

    Article  PubMed  PubMed Central  Google Scholar 

  18. Zhou H, Ye M, Dong J et al (2008) Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis. J Proteome Res 7:3957–3967. doi:10.1021/pr800223m

    Article  CAS  PubMed  Google Scholar 

  19. Imanishi SY, Kochin V, Ferraris SE et al (2007) Reference-facilitated phosphoproteomics: fast and reliable phosphopeptide validation by microLC-ESI-Q-TOF MS/MS. Mol Cell Proteomics 6:1380–1391. doi:10.1074/mcp.M600480-MCP200

    Article  CAS  PubMed  Google Scholar 

  20. McNulty DE, Annan RS (2008) Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol Cell Proteomics 7:971–980. doi:10.1074/mcp.M700543-MCP200

    Article  CAS  PubMed  Google Scholar 

  21. Beausoleil SA, Jedrychowski M, Schwartz D et al (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 101:12130–12135. doi:10.1073/pnas.0404720101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Olsen JV, Blagoev B, Gnad F et al (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127:635–648. doi:10.1016/j.cell.2006.09.026

    Article  CAS  PubMed  Google Scholar 

  23. Gauci S, Helbig AO, Slijper M et al (2009) Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal Chem 81:4493–4501. doi:10.1021/ac9004309

    Article  CAS  PubMed  Google Scholar 

  24. Batth TS, Francavilla C, Olsen JV (2014) Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics. J Proteome Res 13:6176–6186. doi:10.1021/pr500893m

    Article  CAS  PubMed  Google Scholar 

  25. Wiśniewski JR, Nagaraj N, Zougman A et al (2010) Brain phosphoproteome obtained by a FASP-based method reveals plasma membrane protein topology. J Proteome Res 9:3280–3289. doi:10.1021/pr1002214

    Article  PubMed  Google Scholar 

  26. Nühse TS, Stensballe A, Jensen ON, Peck SC (2003) Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol Cell Proteomics 2:1234–1243. doi:10.1074/mcp.T300006-MCP200

    Article  PubMed  Google Scholar 

  27. Ong S-E, Mann M (2005) Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol 1:252–262. doi:10.1038/nchembio736

    Article  CAS  PubMed  Google Scholar 

  28. Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906. doi:10.1038/nprot.2007.261

    Article  CAS  PubMed  Google Scholar 

  29. Weinert BT, Scholz C, Wagner SA et al (2013) Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation. Cell Rep 4:842–851. doi:10.1016/j.celrep.2013.07.024

    Article  CAS  PubMed  Google Scholar 

  30. Wiśniewski JR, Zougman A, Mann M (2009) Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J Proteome Res 8:5674–5678. doi:10.1021/pr900748n

    Article  PubMed  Google Scholar 

  31. Ishihama Y, Rappsilber J, Andersen JS, Mann M (2002) Microcolumns with self-assembled particle frits for proteomics. J Chromatogr A 979:233–239

    Article  CAS  PubMed  Google Scholar 

  32. Michalski A, Damoc E, Hauschild JP et al (2011) Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol Cell Proteomics 10(M111):011015. doi:10.1074/mcp.M111.011015

    PubMed  Google Scholar 

  33. Kelstrup CD, Young C, Lavallee R et al (2012) Optimized fast and sensitive acquisition methods for shotgun proteomics on a quadrupole orbitrap mass spectrometer. J Proteome Res 11:3487–3497. doi:10.1021/pr3000249

    Article  CAS  PubMed  Google Scholar 

  34. Olsen JV, Macek B, Lange O et al (2007) Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods 4:709–712. doi:10.1038/nmeth1060

    Article  CAS  PubMed  Google Scholar 

  35. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372. doi:10.1038/nbt.1511

    Article  CAS  PubMed  Google Scholar 

  36. Tyanova S, Mann M, Cox J (2014) MaxQuant for in-depth analysis of large SILAC datasets. Methods Mol Biol 1188:351–364. doi:10.1007/978-1-4939-1142-4_24

    Article  PubMed  Google Scholar 

  37. Cox J, Neuhauser N, Michalski A et al (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10:1794–1805. doi:10.1021/pr101065j

    Article  CAS  PubMed  Google Scholar 

  38. Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:Article3. doi:10.2202/1544-6115.1027

    PubMed  Google Scholar 

  39. Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98:5116–5121. doi:10.1073/pnas.091062498

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Bendall SC, Hughes C, Stewart MH et al (2008) Prevention of amino acid conversion in SILAC experiments with embryonic stem cells. Mol Cell Proteomics 7:1587–1597. doi:10.1074/mcp.M800113-MCP200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Cox J, Matic I, Hilger M et al (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4:698–705. doi:10.1038/nprot.2009.36

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The research in PB’s group is supported by the German Research Foundation (Emmy Noether Program, BE 5342/1-1 and SFB 1177 on Selective Autophagy) and the Marie Curie Career Integration Grant from the European Commision (grant agreement number: 630763). The research in SAW’s group is supported by the LOEWE program Ubiquitin Networks (Ub-Net) of the State of Hesse (Germany).

Author information

Authors and Affiliations

  1. Institute of Molecular Biology (IMB), Mainz, Germany

    Marina E. Borisova & Petra Beli

  2. Department of Medicine, Hematology/Oncology, Goethe University, Frankfurt, Germany

    Sebastian A. Wagner

Authors
  1. Marina E. Borisova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sebastian A. Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Petra Beli
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Queensland Centre for Clinical Research (UQCCR), University of Queensland, Herston, Queensland, Australia

    Sergei V. Kozlov

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Borisova, M.E., Wagner, S.A., Beli, P. (2017). Mass Spectrometry-Based Proteomics for Quantifying DNA Damage-Induced Phosphorylation. In: Kozlov, S. (eds) ATM Kinase. Methods in Molecular Biology, vol 1599. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6955-5_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6955-5_16

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6953-1

  • Online ISBN: 978-1-4939-6955-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation