LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation
Fig 2
LC-MS-MS assay measures the demethylation activity of FTO in vitro.
(a) (left) LC-MS-MS profiles of nucleosides derived from enzymatic hydrolysis of RNA samples containing m6A upon treatment with the FTO protein. The upper LC-MS-MS profile shows nucleoside A and m6A standards. (Right) Quantitation of m6A in vitro reaction system of FTO and synthetic RNA substrates. (b) (left) LC-MS-MS profiles of nucleosides derived from enzymatic hydrolysis of DNA samples containing 5mdC upon treatment with the FTO proteins. The upper LC-MS-MS profile shows nucleoside 5hmdC, dC and 5mdC standards. (Right) Quantitation of 5mdC in vitro reaction system of FTO and synthetic DNA. (c) FTO catalyzes the demethylation of m6A in a dose- and time-dependent manner. (d) FTO shows no obvious converting 5mdC to 5hmdC in DNA with the increase of the dose of FTO and reaction time. *p < 0.05; N.S.: No Significance. Error bars, mean ± S.E.M. for triplicate experiments.