[Detecting acetylated proteins by affinity chromatography column].
OBJECTIVE
To establish a rapid, relatively quantitative method of detecting acetylated proteins.


METHODS
The proteins of Jurkat cells were acetylated by Trichostatin A (TSA) at different concentrations, then enriched and purified by anti-acetylated lysine antibodies affinity chromatography colum. The components eluted by acid were fixed on the microplate, the levels of acetylated proteins were tested by ELISA, and their components were identified by MALDI-TOF-TOF mass spectrometry. Also the above-mentioned methods were applied to the other three agents (gallic acid, emodin and monoacetylated emodin A).


RESULTS
That 4 × 10(5) Jurkat cells treated with 1 µmol/L TSA produced the optimal acetylated effect, up to 22 acetylated proteins were identified by MALDI-TOF-TOF, of them 15 were acetylated histones. The other three agents also induced acetylation, the relative values of acetylated proteins of Jurkat cells treated with 35.09 µmol/L and 17.54 µmol/L gallic acid were 4.3% and 14.2% respectively; those as of 28.7% and 11.5% treated with 1.47 µmol/L and 2.94 µmol/L emodin; those as of 22.0% and 3.6% treated with 152.91 µmol/L and 30.58 µmol/L monoacetylated emodin A.


CONCLUSIONS
The method based on affinity chromatography colum may be useful for the detection of acetylated proteins, and could be used to screen agents which target to histone deacetylase.
Guangxi Medical University, Nanning, China.
Li
L
Zheng
Yan-ping
YP
Zhong
Hao
H
Xiao
Yi
Y
Zhou
Rong
R
Luo
Hong-tao
HT
Li
Gang
G
Li
Ming
M
Liao
Min
M
He
D000107:Acetylation; D002846:Chromatography, Affinity; D006657:Histones / Q000032:analysis*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D019169:Jurkat Cells; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006657:Histones; D006877:Hydroxamic Acids; C012589:trichostatin A