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

  • Sharon C. Mithoe
  • Frank L. H. Menke
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 

Notes

Acknowledgement

This work was partly financed through Gatsby Charitable Foundation.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.John Innes CentreNorwichUK
  2. 2.ProteomicsThe Sainsbury LaboratoryNorwichUK

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