Detection of Histone H3 Methylation and Acetylation Modifications by Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Epigenetic Drug Evaluation

The changes in epigenetic modifications of histones are one of the important factors in cancer development and metastasis, and the development of epigenetic therapies for cancer treatment has led to epigenetic drug screening as a research focus. In this work, the common methylation and acetylation modifications at the N -terminal of cellular histones H3 were quantified by a liquid chromatography -tandem mass spectrometry (LC -MS/ MS) method, and a throughput assay for screening and assessment of epigenetic drug was established. A total of 39 kinds of modification combinations containing common methylation and acetylation sites of H3 peptides were simultaneously monitored by triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode. The developed method was applied to analyze HepG2 cells exposed for 24 h to 28 kinds of epigenetic drugs that could modulate the level of methylation or acetylation modifications. Results showed that 25 of these drugs, such as deacetylase inhibitors Abexinostat, Valproic acid and AGK7, induced histone H3 modification changes in the exposed cells that were consistent with those reported in the literature, while other modification changes were also detectable. Three of these drugs, including demethylase inhibitors IOX1, GSK-jl and acetyltransferase inhibitor L002, however, induced modification changes different from those reported in the literature. An overall test match rate of 89.3% was achieved. The established LC-MS/MS method could quantitatively analyze histone H3 modification sites and their changes in cells in a high -throughput and highly sensitive manner, and could be applied to the evaluation of epigenetic drugs with known activities, with good specificity and rich modification information, which was expected to provide a new technological tool for screening and evaluation of epigenetically active compounds and exploration of their mechanism of action.