Development of Highly Sensitive Phosphopeptide Enrichment Systems for Quantitative Phosphoproteomics.

Protein phosphorylation/dephosphorylation plays important roles in signal transduction, which regulates many biological processes. Dysfunction of any component may result in a specific disease. Therefore, systemic studies on the regulation mechanism or dynamics of phosphoproteome may help in understanding the mechanism of disease development, and certain components may also emerge as therapeutic targets and lead to the development of new drugs. However, the phosphoproteome analysis is challenging due to their low stoichiometry of modification. Detection of phosphopeptides may only be effective when a proper enrichment method was used. Based on our experience in handling a great variety of samples, we realized that the online multi-dimensional separation is an effective method for peptide separation. Therefore, we planned to establish high performance phosphopeptide analysis platforms. The concept is based on an in-line dilution design which connecting different dimension of separations. This design solved the possible solvent incompatibility problem between separations. The online MDLC system will not only possess excellent separation performance but also maximize sample recovery. In this two-year proposal, we will establish three systems which can handle different scale of samples. They are (1) TiO2/RP-2DLC, (2) SAX/TiO2/RP-3DLC and (3) COFRADIC (combined fractional diagonal chromatography) system. Standard proteins and a cell model will be used to evaluate the performance of these systems. We hope the established platforms can be used to analyze trace amount of samples such as laser capture microdissected tissue samples.

Project ID:PC10001-0976
External Project ID:NSC99-2320-B182-011-MY2