Subject Area物理化学
Development of Technology and Methods for Large-scale Phosphoproteome Analysis
Zou HF(邹汉法); Ye ML(叶明亮); Jiang XN(江新宁); Han GH(韩广辉); Wang FJ(王方军)
Conference Name2010 International Chemical Congress of Pacific Basin Societies
Conference Date2010-12-15
Conference Place美国
Alternative Title大尺度磷酸化蛋白质组分析新方法和新技术
Funding Organization2010年泛太平洋国际化学会议组委会
AbstractThe 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. A new reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach was developed for multidimensional separation of phosphopeptides. In this approach, a large number of fractions were collected from the first dimensional RPLC separation at high pH. And then these fractions were pooled every two fractions with equal time interval, one from the early eluted section and another one from the later eluted section. The pooled fractions were finally submitted to RPLC−tandem mass spectrometry (MS/MS) analysis at low pH. It was found the resulting 2D separation was highly orthogonal and yielded more than 30% phosphopeptide identifications over the conventional RP-RPLC approach. 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. A software suit with classification filtering criteria 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. Finally the developed methods and technology were integrated for large-scale phosphoproteome analysis of human liver, which leads to identification of 10644 unique phosphopeptides corresponding to 3149 unique phosphoproteins and 9995 unique phosphosites, respectively.
Document Type会议论文
Corresponding AuthorZou HF(邹汉法)
Recommended Citation
GB/T 7714
Zou HF,Ye ML,Jiang XN,et al. Development of Technology and Methods for Large-scale Phosphoproteome Analysis[C],2010:59/2.
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