Affinity Purification Followed by Liquid Chromatography–Tandem Mass Spectrometry to Identify Proteins Interacting with ABA Signaling Components

Fumiyuki Soma, Fuminori Takahashi, Kazuo Shinozaki, Kazuko Yamaguchi-Shinozaki

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

Abscisic acid (ABA) is a key phytohormone involved in plant development, seed germination and responses to osmotic stresses, such as drought and high salinity. SNF1-related protein kinases (SnRK2s) play important roles in ABA-dependent and ABA-independent osmotic stress signaling. SnRK2s phosphorylate transcription factors and ion channels in response to ABA or osmotic stress to induce the expression of stress-responsive genes and stomatal closure, respectively, to confer osmotic stress tolerance. The activity of SnRK2s is directly or indirectly regulated by several protein factors. Identification of downstream substrates or upstream regulators of SnRK2s is very useful for elucidating protein components that regulate ABA and osmotic stress signaling. Here, we describe the use of affinity purification by coimmunoprecipitation and liquid chromatography–tandem mass spectrometry to identify protein complexes involved in ABA and osmotic stress signaling in plants. We previously identified several protein factors that regulate ABA and osmotic stress signaling by using this method.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages181-189
Number of pages9
DOIs
Publication statusPublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2462
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Coimmunoprecipitation
  • GFP-tagged protein
  • In-gel digestion
  • LC-MS/MS analysis
  • Peptide purification

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