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Identification of MAPK Substrates Using Quantitative Phosphoproteomics

  • Tong Zhang
  • Jacqueline D. Schneider
  • Ning Zhu
  • Sixue ChenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)

Abstract

Activation of mitogen-activated protein kinases (MAPKs) under diverse biotic and abiotic factors and identification of an array of downstream MAPK target proteins are hot topics in plant signal transduction. Through interactions with a plethora of substrate proteins, MAPK cascades regulate many physiological processes in the course of plant growth, development, and response to environmental factors. Identification and quantification of potential MAPK substrates are essential, but have been technically challenging. With the recent advancement in phosphoproteomics, here we describe a method that couples metal dioxide for phosphopeptide enrichment with tandem mass tags (TMT) mass spectrometry (MS) for large-scale MAPK substrate identification and quantification. We have applied this method to a transient expression system carrying a wild type (WT) and a constitutive active (CA) version of a MAPK. This combination of genetically engineered MAPKs and phosphoproteomics provides a high-throughput, unbiased analysis of MAPK-triggered phosphorylation changes on the proteome scale. Therefore, it is a robust method for identifying potential MAPK substrates and should be applicable in the study of other kinase cascades in plants as well as in other organisms.

Key words

MAPK MAPK substrate Signal transduction Phosphoproteomics TMT 

Notes

Acknowledgments

This research was supported by grants from the National Science Foundation (MCB 0818051 and MCB 1412547) to S. Chen.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Tong Zhang
    • 1
    • 2
  • Jacqueline D. Schneider
    • 1
    • 3
  • Ning Zhu
    • 1
  • Sixue Chen
    • 1
    • 2
    • 4
    • 5
    Email author
  1. 1.Department of BiologyUniversity of FloridaGainesvilleUSA
  2. 2.Genetics InstituteUniversity of FloridaGainesvilleUSA
  3. 3.Department of Chemical EngineeringUniversity of FloridaGainesvilleUSA
  4. 4.Plant Molecular and Cellular Biology ProgramUniversity of FloridaGainesvilleUSA
  5. 5.Interdisciplinary Center for Biotechnology ResearchUniversity of FloridaGainesvilleUSA

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