Skip to main content

Phosphopeptide Immuno-Affinity Enrichment to Enhance Detection of Tyrosine Phosphorylation in Plants

  • Protocol

Part of the Methods in Molecular Biology book series (MIMB,volume 1306)

Abstract

Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems, but the relative contribution of Tyr phosphorylation to plant signal transduction has, until recently, remained an open question. One of the major issues hampering the analysis is the low abundance of Tyr phosphorylation and therefore underrepresentation in most mass spec-based proteomic studies. Here, we describe a working approach to selectively enrich Tyr-phosphorylated peptides from complex plant tissue samples. We describe a detailed protocol that is based on immuno-affinity enrichment step using an anti-phospho-tyrosine (pTyr)-specific antibody. This single enrichment strategy effectively enriches pTyr-containing peptides from complex total plant cell extracts, which can be measured by LC-MS/MS without further fractionation or enrichment.

Key words

  • Immuno-affinity enrichment
  • Tyrosine
  • Posttranslational modifications
  • Phosphorylation
  • Arabidopsis
  • Metabolic labeling
  • Mass spectrometry

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-2648-0_10
  • Chapter length: 12 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   89.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-2648-0
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   119.00
Price excludes VAT (USA)
Hardcover Book
USD   169.99
Price excludes VAT (USA)

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

References

  1. Boersema PJ, Foong LY, Ding VM, Lemeer S, van Breukelen B, Philp R, Boekhorst J, Snel B, den Hertog J, Choo AB, Heck AJ (2010) In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling. Mol Cell Proteomics 9(1):84–99

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  2. Ding VM, Boersema PJ, Foong LY, Preisinger C, Koh G, Natarajan S, Lee DY, Boekhorst J, Snel B, Lemeer S, Heck AJ, Choo A (2011) Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells. PLoS One 6(3):e17538

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  3. Del Rosario AM, White FM (2010) Quantifying oncogenic phosphotyrosine signaling networks through systems biology. Curr Opin Genet Dev 20(1):23–30. doi:10.1016/j.gde.2009.12.005

    CrossRef  PubMed Central  PubMed  Google Scholar 

  4. Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141(7): 1117–1134

    Google Scholar 

  5. Macho AP, Schwessinger B, Ntoukakis V, Brutus A, Segonzac C, Roy S, Kadota Y, Oh MH, Sklenar J, Derbyshire P, Lozano-Duran R, Malinovsky FG, Monaghan J, Menke FL, Huber SC, He SY, Zipfel C (2014) A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation. Science 343(6178):1509–1512

    CrossRef  CAS  PubMed  Google Scholar 

  6. Mithoe SC, Menke FLH (2011) Phosphoproteomics perspective on plant signal transduction and tyrosine phosphorylation. Phytochemistry 72(10):997–1006

    CrossRef  CAS  PubMed  Google Scholar 

  7. de la Fuente van Bentem S, Hirt H (2009) Protein tyrosine phosphorylation in plants: More abundant than expected? Trends Plant Sci 14(2):71–76. doi:10.1016/j.tplants.2008.11.003

    CrossRef  CAS  PubMed  Google Scholar 

  8. Benschop JJ, Mohammed S, O’Flaherty M, Heck AJR, Slijper M, Menke FLH (2007) Quantitative Phosphoproteomics of Early Elicitor Signaling in Arabidopsis. Mol Cell Proteomics 6(7):1198–1214

    CrossRef  CAS  PubMed  Google Scholar 

  9. Nuhse TS, Stensballe A, Jensen ON, Peck SC (2004) Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16(9):2394–2405

    CrossRef  PubMed Central  PubMed  Google Scholar 

  10. de la Fuente van Bentem S, Anrather D, Dohnal I, Roitinger E, Csaszar E, Joore J, Buijnink J, Carreri A, Forzani C, Lorkovic ZJ, Barta A, Lecourieux D, Verhounig A, Jonak C, Hirt H (2008) Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis. J Proteome Res 7(6):2458–2470

    CrossRef  CAS  PubMed  Google Scholar 

  11. Sugiyama N, Nakagami H, Mochida K, Daudi A, Tomita M, Shirasu K, Ishihama Y (2008) Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis. Mol Syst Biol 4

    Google Scholar 

  12. Mithoe SC, Boersema PJ, Berke L, Snel B, Heck AJ, Menke FL (2012) Targeted quantitative phosphoproteomics approach for the detection of phospho-tyrosine signaling in plants. J Proteome Res 11(1):438–448

    CrossRef  CAS  PubMed  Google Scholar 

  13. Grimsrud PA, den Os D, Wenger CD, Swaney DL, Schwartz D, Sussman MR, Ane JM, Coon JJ (2010) Large-scale phosphoprotein analysis in Medicago truncatula roots provides insight into in vivo kinase activity in legumes. Plant Physiol 152(1):19–28. doi:10.1104/pp.109.149625

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  14. Zhang Y, Wolf-Yadlin A, Ross PL, Pappin DJ, Rush J, Lauffenburger DA, White FM (2005) Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules. Mol Cell Proteomics 4(9):1240–1250

    CrossRef  CAS  PubMed  Google Scholar 

  15. Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, Nardone J, Lee K, Reeves C, Li Y, Hu Y, Tan Z, Stokes M, Sullivan L, Mitchell J, Wetzel R, Macneill J, Ren JM, Yuan J, Bakalarski CE, Villen J, Kornhauser JM, Smith B, Li D, Zhou X, Gygi SP, Gu TL, Polakiewicz RD, Rush J, Comb MJ (2007) Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131(6):1190–1203

    CrossRef  CAS  PubMed  Google Scholar 

  16. Oh MH, Wang X, Kota U, Goshe MB, Clouse SD, Huber SC (2009) Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis. Proc Natl Acad Sci U S A 106(2):658–663

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  17. Thingholm TE, Jensen ON, Larsen MR (2009) Analytical strategies for phosphoproteomics. Proteomics 9(6):1451–1468

    CrossRef  CAS  PubMed  Google Scholar 

  18. Bindschedler LV, Palmblad M, Cramer R (2008) Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study. Phytochemistry 69(10):1962–1972

    CrossRef  CAS  PubMed  Google Scholar 

  19. Lind SB, Artemenko KA, Pettersson U (2012) A strategy for identification of protein tyrosine phosphorylation. Methods 56(2):275–283

    CrossRef  CAS  PubMed  Google Scholar 

Download references

Acknowledgement

This work was partly financed through Gatsby Charitable Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Frank L. H. Menke .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Mithoe, S.C., Menke, F.L.H. (2015). Phosphopeptide Immuno-Affinity Enrichment to Enhance Detection of Tyrosine Phosphorylation in Plants. In: Schulze, W. (eds) Plant Phosphoproteomics. Methods in Molecular Biology, vol 1306. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2648-0_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2648-0_10

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2647-3

  • Online ISBN: 978-1-4939-2648-0

  • eBook Packages: Springer Protocols