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Abstract No.: TuP-206
Session: Proteomics: New Methods
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

High-throughput Quantitative Mass Analysis of Phosphoproteome by Affinity Enrichment and Chemical Labeling

Yeong Hee Ahn1, Kun Cho1, Jin Young Kim1, Kyung-Hoon Kwon1, Jong Shin Yoo1

1 Korea Basic Science Institute, Daejon, Korea

Correspondence address: Yeong Hee Ahn, Korea Basic Science Institute, 52 Eeoeun-Dong, Yusung-Gu, Daejon, 305-333 Korea.

Keywords: Affinity, Metal; Labeling, Isotope; Post-translational Modification; Quantitative Analysis.

Novel aspect: Phosphoselective enrichment and site-specific labeling for quantitative phosphoproteome analysis.

 

Protein phosphorylation is of particular interest since it is one of the most common protein modification, and playing a central role in biological process like cell-regulation and signaling etc. Therefore robust methods for the characterization of phosphorylated proteins are essential for further understanding the mechanism of phosphorylation and its biological action. One of subjects to be improved further in analytical proteomic field is efficient enrichment and high sensitive analysis for phosphoproteome. Immobilized metal affinity chromatography (IMAC) is a well known phosphoproteome enrichment technique. Site-specific chemical tagging for phosphorylated site is also introduced for high sensitive detection and quantification of phosphoproteome. The efficient coupling of two methods, the phospho-selective enrichment and the site-specific chemical labeling, provides a powerful tool for practical phosphoproteome analysis. Phosphopeptide is selectively enriched on metal affinity resin under acidic condition. After thorough washing of nonphosphopeptide, enriched phosphopeptide is directly subjected to site-specific labeling by a chemical tag showing high proton affinity. The labeled peptide shows greatly increased mass sensitivity and gives some unique fragment ion peaks in tandem mass spectra, which enables high sensitive mass analysis of phosphoproteome. Stable isotope-coded mass tag is also useful for relative quantitation of phosphoproteome. It is also introduced the relative quantitation method for peptides enriched by metal and labeled with chemical tag.