Skip to main content
SpringerLink
Log in
Book cover

Systems Chemical Biology pp 141–152Cite as

Examining Cellular Responses to Kinase Drug Inhibition Through Phosphoproteome Mapping of Substrates

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1888))

Abstract

This chapter provides a detailed description for mass spectrometry-based phosphoproteomics analysis. We describe sample preparation, phosphopeptide enrichment, mass spectrometry acquisition, label-free data analysis and statistical analysis. This technique can be employed to characterize cell signaling networks, and is particularly useful to monitor cellular responses to kinase drug inhibitors.

This is a preview of subscription content, 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
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Hunter T (2000) Signaling—2000 and beyond. Cell 100(1):113–127

    Article  CAS  Google Scholar 

  2. Smith CC, Wang Q, Chin CS, Salerno S, Damon LE, Levis MJ, Perl AE, Travers KJ, Wang S, Hunt JP, Zarrinkar PP, Schadt EE, Kasarskis A, Kuriyan J, Shah NP (2012) Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature 485(7397):260–263. https://doi.org/10.1038/nature11016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW (1997) Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275(5307):1787–1790

    Article  CAS  Google Scholar 

  4. Reimand J, Wagih O, Bader GD (2013) The mutational landscape of phosphorylation signaling in cancer. Sci Rep 3:2651. https://doi.org/10.1038/srep02651

    Article  PubMed  PubMed Central  Google Scholar 

  5. Radivojac P, Baenziger PH, Kann MG, Mort ME, Hahn MW, Mooney SD (2008) Gain and loss of phosphorylation sites in human cancer. Bioinformatics 24(16):i241–i247. https://doi.org/10.1093/bioinformatics/btn267

    Article  PubMed  PubMed Central  Google Scholar 

  6. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, Dummer R, Garbe C, Testori A, Maio M, Hogg D, Lorigan P, Lebbe C, Jouary T, Schadendorf D, Ribas A, O'Day SJ, Sosman JA, Kirkwood JM, Eggermont AM, Dreno B, Nolop K, Li J, Nelson B, Hou J, Lee RJ, Flaherty KT, McArthur GA, Group B-S (2011) Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 364(26):2507–2516. https://doi.org/10.1056/NEJMoa1103782

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tiacci E, Trifonov V, Schiavoni G, Holmes A, Kern W, Martelli MP, Pucciarini A, Bigerna B, Pacini R, Wells VA, Sportoletti P, Pettirossi V, Mannucci R, Elliott O, Liso A, Ambrosetti A, Pulsoni A, Forconi F, Trentin L, Semenzato G, Inghirami G, Capponi M, Di Raimondo F, Patti C, Arcaini L, Musto P, Pileri S, Haferlach C, Schnittger S, Pizzolo G, Foa R, Farinelli L, Haferlach T, Pasqualucci L, Rabadan R, Falini B (2011) BRAF mutations in hairy-cell leukemia. N Engl J Med 364(24):2305–2315. https://doi.org/10.1056/NEJMoa1014209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. van Veelen W, Le NH, Helvensteijn W, Blonden L, Theeuwes M, Bakker ER, Franken PF, van Gurp L, Meijlink F, van der Valk MA, Kuipers EJ, Fodde R, Smits R (2011) beta-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis. Gut 60(9):1204–1212. https://doi.org/10.1136/gut.2010.233460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Mann M, Hendrickson RC, Pandey A (2001) Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem 70:437–473. https://doi.org/10.1146/annurev.biochem.70.1.437

    Article  CAS  PubMed  Google Scholar 

  10. Kuster B, Mortensen P, Andersen JS, Mann M (2001) Mass spectrometry allows direct identification of proteins in large genomes. Proteomics 1(5):641–650. https://doi.org/10.1002/1615-9861(200104)1:5<641::AID-PROT641>3.0.CO;2-R

    Article  CAS  PubMed  Google Scholar 

  11. Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422(6928):198–207. https://doi.org/10.1038/nature01511

    Article  CAS  PubMed  Google Scholar 

  12. Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jørgensen TJ (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4(7):873–886

    Article  CAS  Google Scholar 

  13. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1(5):376–386

    Article  CAS  Google Scholar 

  14. Mueller LN, Brusniak M-Y, Mani D, Aebersold R (2008) An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data. J Proteome Res 7(01):51–61

    Article  CAS  Google Scholar 

  15. Eyrich B, Sickmann A, Zahedi RP (2011) Catch me if you can: Mass spectrometry-based phosphoproteomics and quantification strategies. Proteomics 11(4):554–570

    Article  CAS  Google Scholar 

  16. Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13(9):2513–2526

    Article  CAS  Google Scholar 

  17. Parker R, Clifton-Bligh R, Molloy MP (2014) Phosphoproteomics of MAPK inhibition in BRAF-mutated cells and a role for the lethal synergism of dual BRAF and CK2 inhibition. Mol Cancer Ther 13(7):1894–1906

    Article  CAS  Google Scholar 

  18. Casado P, Hijazi M, Britton D, Cutillas PR (2016) Impact of phosphoproteomics in the translation of kinase targeted therapies. Proteomics. https://doi.org/10.1002/pmic.201600235

  19. Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016) The Perseus computational platform for comprehensive analysis of (prote) omics data. Nat Methods 13(9):731–740

    Article  CAS  Google Scholar 

  20. Rogers LD, Fang Y, Foster LJ (2010) An integrated global strategy for cell lysis, fractionation, enrichment and mass spectrometric analysis of phosphorylated peptides. Mol BioSyst 6(5):822–829

    Article  CAS  Google Scholar 

  21. Zhou J, Zhou T, Cao R, Liu Z, Shen J, Chen P, Wang X, Liang S (2006) Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane. J Proteome Res 5(10):2547–2553

    Article  CAS  Google Scholar 

  22. Sugiyama N, Masuda T, Shinoda K, Nakamura A, Tomita M, Ishihama Y (2007) Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol Cell Proteomics 6(6):1103–1109

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Sciences, ARC Training Centre for Molecular Technology in the Food Industry, Macquarie University, Sydney, Australia

    Daniel Bucio-Noble, Crystal Semaan & Mark P. Molloy

  2. Australian Proteome Analysis Facility, Macquarie University, Sydney, Australia

    Mark P. Molloy

  3. Bowel Cancer and Biomarker Research, Kolling Institute, The University of Sydney, Sydney, Australia

    Mark P. Molloy

Authors
  1. Daniel Bucio-Noble
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Crystal Semaan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark P. Molloy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark P. Molloy .

Editor information

Editors and Affiliations

  1. Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Slava Ziegler

  2. Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Herbert Waldmann

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Bucio-Noble, D., Semaan, C., Molloy, M.P. (2019). Examining Cellular Responses to Kinase Drug Inhibition Through Phosphoproteome Mapping of Substrates. In: Ziegler, S., Waldmann, H. (eds) Systems Chemical Biology. Methods in Molecular Biology, vol 1888. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8891-4_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8891-4_8

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8890-7

  • Online ISBN: 978-1-4939-8891-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation