Enrichment and Analysis of Phosphopeptides Shaved from Membrane Fraction of Salt-stressed Arabidopsis

in International Symposium (poster summary), 國際研討會(摘要海報發表)
標題Enrichment and Analysis of Phosphopeptides Shaved from Membrane Fraction of Salt-stressed Arabidopsis
AuthorsLiao Ming-Huei, 徐睿良, & Cindy Lee 李欣蒂
出版日期Oct 5 2009 12:0

Under salt stress condition, plants respond by turning on phosphorylating cascade. Membrane is the site where key phosphorylation events occur. Nevertheless, so far limited phosphorylation sites in response to salt stress in plants have been reported. In the present work, the salt stress-stimulated protein phosphorylation of membrane proteins in Arabidopsis was investigated. Membrane fractions from Control, 200 mM or 400 mM salt-treated Arabidopsis suspension plants were isolated, followed by protease shaving with the assistance of 60% methanol and enrichment by Zirconium ion-charged magnetic beads. Through the organic solvent promotion, the membrane proteins were prone to be dissolved in this aqueous-organic solvent system and facilitated to the downstream trypsin digestion. The phosphopeptides from different samples were enriched by Zr4+-IMAC (immobilized metal ion affinity chromatography) magnetic beads in parallel within five minutes. Through this simple procedure, we identified 17 phosphopeptides from 15 membrane proteins in Arabidopsis. These proteins include a sugar transporter, AHA1, and aquaporins. A phosphorylation site of AHA1 was identified in 200 mM salt-treated plants. A phosphorylation site of a sugar transporter was identified both in 200 and 400 mM slat-treated plants. In this study, we successfully identify phosphorylation sites from membranes of specific abiotic-stressed plants by organic solvent-assisted trypsin digestion followed by Zr4+-IMAC enrichment. In addition, we also identified distinct phosphorylation sites between different salt stress conditions. Further study such as more comprehensive mapping of phosphorylation sites and quantitative analysis of protein phosphorylation with and without salt stress are in progress.

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