Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach

Sun Jae Kwon, Eun Young Choi, Jong Bok Seo, Ohkmae Kim

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bis-phosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDITOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach.

Original languageEnglish
Pages (from-to)268-275
Number of pages8
JournalMolecules and Cells
Volume24
Issue number2
Publication statusPublished - 2007 Oct 31

Fingerprint

Biotin
Arabidopsis
Phosphoproteins
Proteins
Phosphates
Phosphopeptides
Oxygenases
Avidin
Electrophoresis, Gel, Two-Dimensional
Threonine
Seedlings
Affinity Chromatography
Serine
Signal Transduction
Mass Spectrometry
Lasers
Phosphorylation

Keywords

  • Arabidopsis
  • Biotin-tagging
  • Mass spectrometry
  • Phosphoproteome
  • Protein phosphorylation

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach. / Kwon, Sun Jae; Choi, Eun Young; Seo, Jong Bok; Kim, Ohkmae.

In: Molecules and Cells, Vol. 24, No. 2, 31.10.2007, p. 268-275.

Research output: Contribution to journalArticle

Kwon, Sun Jae ; Choi, Eun Young ; Seo, Jong Bok ; Kim, Ohkmae. / Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach. In: Molecules and Cells. 2007 ; Vol. 24, No. 2. pp. 268-275.
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