? The goal of this research is to develop a proteomics approach to isolate and quantify ubiquitinated proteome from tumor tissues based on high resolution mass spectrometry. Ubiquitin modification regulates a variety of cellular events in eukaryotic cells by covalently attaching ubiquitin to target substrates. The dysregulation of ubiquitin pathways has been involved in the pathogenesis of many types of cancers. However, there is no reliable method to globally analyze ubiquitinated proteins in cancer samples. Recently, we have made progress in developing novel mass spectrometry-based technologies to analyze ubiquitinated proteome in yeast. Here we propose to develop a generic method to capture and quantify ubiquitinated proteome from mammalian cells. The approach will utilize affinity chromatography composed of ubiquitin-binding domains. An array of fusion proteins with various ubiquitin-binding domains will be examined to optimize the binding affinity in order to improve the method with respect to the yield and purity of ubiquitin substrates. The isolated ubiquitinated proteins will be further analyzed by mass spectrometry for the determination of protein identity and ubiquitination sites. Quantitative mass spectrometry strategies will be implemented to investigate the dynamics of ubiquitinated proteome. Once established, the method will be highly useful for profiling ubiquitinated proteome in mammalian samples including clinical tumor tissues. ? ? ?
| Xu, Ping; Duong, Duc M; Seyfried, Nicholas T et al. (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137:133-45 |
| Xu, Ping; Duong, Duc M; Peng, Junmin (2009) Systematical optimization of reverse-phase chromatography for shotgun proteomics. J Proteome Res 8:3944-50 |
| Xu, Ping; Peng, Junmin (2008) Characterization of polyubiquitin chain structure by middle-down mass spectrometry. Anal Chem 80:3438-44 |
| Seyfried, Nicholas T; Xu, Ping; Duong, Duc M et al. (2008) Systematic approach for validating the ubiquitinated proteome. Anal Chem 80:4161-9 |
| Peng, Junmin (2008) Evaluation of proteomic strategies for analyzing ubiquitinated proteins. BMB Rep 41:177-83 |
