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Plant Phosphoproteomics pp 135–146Cite as

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

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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

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  • DOI: 10.1007/978-1-4939-2648-0_10
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Acknowledgement

This work was partly financed through Gatsby Charitable Foundation.

Author information

Authors and Affiliations

  1. John Innes Centre, Norwich, UK

    Sharon C. Mithoe

  2. Proteomics, The Sainsbury Laboratory, Colney Lane, Norwich, NR4 7UH, UK

    Frank L. H. Menke

Authors
  1. Sharon C. Mithoe
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  2. Frank L. H. Menke
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Corresponding author

Correspondence to Frank L. H. Menke .

Editor information

Editors and Affiliations

  1. Department of Plant Systems Biology, Universität Hohenheim, Stuttgart, Germany

    Waltraud X. Schulze

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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

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  • 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

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