Protein Turn-over In Cell Cycle Characterized By Quantitative Proteomics

Cell division is the basis of development, growth, homeostasis and function of multicellular organism. The mechanism of cell cycle regulation, one of the most important fields of life science, is well-organized and highly complex. Due to the complication of mammalian cells and the limitation of current research techniques, our understanding of the process is still incomplete. To identify novel cell cycle regulators as well as integrate the networks involved in cell cycle to a global view are especially urgent. Here, we systematically profile the proteome turnover during cell cycle by emerging SILAC-basic quantitative proteomics techniques hoping to open up new dimension of cell cycle research.AIM:Our goals are to globally monitor the dynamic of proteome turnover during cell cycle by quantitative proteomics and link the patterns of protein turnover to functions by bioinformatics analysis.Methods:We have developed a robust protocol for synchronizing cells at phase of G1/S,S, G2, M and G1 at high synchronization index in a single consecutive cell cycle by employing the combination of double thymidine block, Nocodazole and mitotic shake-off. Protein turnover is monitored by SILAC-basic quantitative proteomics. Data is analyzed by bioinformatics tools including MaxQuant, GOstat, Cytoscape, and etc.Results:We have succeeding in collecting cells at phase of G1/S, S, G2, M and G1 at high synchronization index up to 90% in a single consecutive cell cycle, and the results were validated by flow cytometry at DNA level as well as Western blot at protein level. GO enrichment showed that the patterns of protein turnover are closed related to their functions. Furthermore, analysis of protein interaction indicated that a group of cell adhesion proteins which renew quickly are probably involved in cell morphology change during cell cycle.Conclusion:Our study has revealed the close relation between protein turnover patterns with cell cycle process. Hence, globally characterize proteome turnover during cell cycle by SILAC-basic quantitative proteomics will shed new light on the old questions.