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Isolation of the Arabidopsis Phosphoproteome Using a Biotin-tagging Approach  

Kwon, Sun Jae (School of Life Sciences and Biotechnology, Korea University)
Choi, Eun Young (School of Life Sciences and Biotechnology, Korea University)
Seo, Jong Bok (Seoul Branch, Korea Basic Science Institute)
Park, Ohkmae K. (School of Life Sciences and Biotechnology, Korea University)
Publication Information
Molecules and Cells / v.24, no.2, 2007 , pp. 268-275 More about this Journal
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-bisphosphate 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 (MALDI-TOF 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.
Keywords
Arabidopsis; Biotin-Tagging; Mass Spectrometry; Phosphoproteome; Protein Phosphorylation;
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Times Cited By KSCI : 1  (Citation Analysis)
Times Cited By Web Of Science : 7  (Related Records In Web of Science)
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