Study Of Detecting Acetylated Proteins By Affinity Chromatography Column

There was close relationship between protein acetylation and the tumor development. The state of Acetylation depended on the activity competetion between histone acetyltransferase (HATs) and histone deacetylase(HDACs).Lots of experiences shows that HDACs inhibitor could increase the level of acetylation of histones in chromatin causing the activation and expression of specific genes, accordingly leading to the end differentiation and apoptosis of cancer cells.Objective:To establish a rapid, relatively quantitative method for detecting acetylated proteins, and to explore its applications.Method:Different number of Jurkat cells were treated with different concentration of histone deacetylase inhibitor Trichostatin A(TSA) at different time periods. After enrichment and purification with anti-acetylated lysine antibodies affinity chromatography column and then elution by acid, the levels of acetylated protein of Jurkat cells were tested by ELISA; The eluted components were identified by MALDI-TOF-TOF mass spectrometry. A preliminary study on the change of levels of acetylated proteins of Jurkat cells treated with3different traditional medicine monomer, including gallic acid、Emodin and mono acetylated Emodin A, was performed by the method.Result:The relative amount of total acetylated protein of Jurkat cells reached the highest level after4×105number of cells treated with1μmol/L of TSA for24h.22acetylated proteins were identified by MALDI-TOF-TOF in eluent among them15were acetylated histones; Compared with negative control group(without drug), the degree of acetylation of protein of each treatment group was increased, and that of1.47μmol/L of gallic acid group was increased most remarkedly(P<0.01).Conclusion:The method was initially established to enricr、purify and detect the total acetylated proteins in living cells. It might be a fast, simple way for screening anticancer drugs which targeted to deacetylase.