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

  • Daniel Bucio-Noble
  • Crystal Semaan
  • Mark P. MolloyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1888)


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.

Key words

Phosphoproteomics Phosphopeptide enrichment Mass spectrometry Label-free 


  1. 1.
    Hunter T (2000) Signaling—2000 and beyond. Cell 100(1):113–127CrossRefGoogle Scholar
  2. 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. Scholar
  3. 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):1787–1790CrossRefGoogle Scholar
  4. 4.
    Reimand J, Wagih O, Bader GD (2013) The mutational landscape of phosphorylation signaling in cancer. Sci Rep 3:2651. Scholar
  5. 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. Scholar
  6. 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. Scholar
  7. 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. Scholar
  8. 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. Scholar
  9. 9.
    Mann M, Hendrickson RC, Pandey A (2001) Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem 70:437–473. Scholar
  10. 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.<641::AID-PROT641>3.0.CO;2-RCrossRefPubMedGoogle Scholar
  11. 11.
    Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422(6928):198–207. Scholar
  12. 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):873–886CrossRefGoogle Scholar
  13. 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):376–386CrossRefGoogle Scholar
  14. 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):51–61CrossRefGoogle Scholar
  15. 15.
    Eyrich B, Sickmann A, Zahedi RP (2011) Catch me if you can: Mass spectrometry-based phosphoproteomics and quantification strategies. Proteomics 11(4):554–570CrossRefGoogle Scholar
  16. 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):2513–2526CrossRefGoogle Scholar
  17. 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):1894–1906CrossRefGoogle Scholar
  18. 18.
    Casado P, Hijazi M, Britton D, Cutillas PR (2016) Impact of phosphoproteomics in the translation of kinase targeted therapies. Proteomics.
  19. 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):731–740CrossRefGoogle Scholar
  20. 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):822–829CrossRefGoogle Scholar
  21. 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):2547–2553CrossRefGoogle Scholar
  22. 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):1103–1109CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Daniel Bucio-Noble
    • 1
  • Crystal Semaan
    • 1
  • Mark P. Molloy
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Molecular Sciences, ARC Training Centre for Molecular Technology in the Food IndustryMacquarie UniversitySydneyAustralia
  2. 2.Australian Proteome Analysis FacilityMacquarie UniversitySydneyAustralia
  3. 3.Bowel Cancer and Biomarker Research, Kolling InstituteThe University of SydneySydneyAustralia

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