Study Of Protein Phosphorylation Based On Proteomic Analysis Using 2-DE

Phosphorylation is a kind of important protein post-translational modification, the study of protein phosphorylation is of great significance to understand the essence of life. Based on traditional proteomic study, this research explore the methods for phosphoprotein extraction, enrichment and detection from rice leaves, finds a way to predict protein phosphorylation based on protein identification with MS and a strategy to detect phosphorylation from protein spots, offers a feasible strategy to study the phosphorylation of differential displayed protein spots after proteomic study base on 2-DE.In this study, it is proved that traditional TCA/Acetone method and phenol extraction method is not efficient enough to extract phosphoprotein from rice leaves. After precipitate Rubisco from rice leaf total protein using ammonium sulfate, more protein spots are detected on 2-DE map, which may increase the efficiency of phosphoprotein extraction with low amount.This research finds a method to enrich phosphoproteins dissolved in solutions with high concentration of urea, which is used to enrich phosphoproteins of rice leaves, and detect 8 pairs of proteins whose amounts are changed after phosphatase treatment, using the method of phosphatase treatment.Besides, this study offers a method to predict protein phosphorylation based on protein identification with MS, which is used to predict the phosphorylation of differential displayed protein spots during cotton ovule development. From 50 differential displayed protein spots, 799 potential phosphorylated sites are predicted with the ranking of phosphorylation possibility. Based on phosphorylated site prediction, all the 50 protein spots are classified according to their phosphorylation level.Finally, the study offer a method named as phosphatase treatment method to detect phosphorylation for those protein spots excised from 2-DE gels. Proved by standard sample, this method can effectively distinguish phosphorylated proteins from unphosphorylated protein. Using phosphatase treatment method, 9 protein spots with electrophoretic moving ability shift after phosphatase treatment are detected from 50 differential displayed protein spots during cotton ovule development.