Keynote Speaker: Dr. Hanfa Zou Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China Sample preparation and data processes for phosphoproteome analysis The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. A new type of the immobilized metal ion affinity chromatography (IMAC) through the chelating interaction between phosphate groups on the polymer and Zr4+ and Ti4+ (Zr4+- and Ti4+-IMAC) has been developed for enriching phosphopeptides. We also compared Zr4+- and Ti4+-IMAC to other enrichment methods including Fe3+-IMAC, TiO2 and ZrO2, and demonstrate superior selectivity and efficiency of Zr4+- and Ti4+-IMAC for the isolation and enrichment of phosphopeptides. Furthermore, a new approach was established by integration of the enrichment of phosphopeptides with Ti4+-IMAC and fractionation of the enriched phosphopeptides with strong anion-exchange (SAX) capillary trap column for phosphoproteome analysis. It was observed and the total number of the identified phosphopeptides increased 105.6% by comparing to conventional method with directly loading Ti-IMAC enriched fraction onto C18 trap column for capillary liquid chromatography-tandem mass spectrometry analysis. During phosphoproteome analysis by capillary LC-MS/MS technique, manual checking is commonly employed to validate the phosphopeptide identifications from database searching of tandem mass spectra. It is very time-consuming and labor intensive as the number of phosphopeptide identifications increases greatly. An automatic validation approach with software tool was developed for phosphopeptide identification by combining consecutive stage mass spectrometry data and the target-decoy database searching strategy. It was demonstrated that the determined FDR can precisely reflect the actual FDR without any manual validation stage.