Systematic Study Epigenetic Metabolome In Biological Samples By Targeted Metabolomic Method

OBJECTIVE: Epigenetics related metabolites are direct substrates or cofactors,which are involved in epigenetic modifications in enzymatic and non-enzymatic reactions.They determine the way and intensity of epigenetic modifications.We refer to these collections of epigenetic related metabolites as epigenetic metabolome.Studies have found that changes in the epigenetic related metabolites were observed in specific disease states,supplement or limit the intake of certain epigenetic-related metabolites can affect the progression of those diseases,therefore,the epigenetic metabolome is closely related to the diseases.At present,the epigenetic researches focuse on studying a single modification directly,there have been no studies specifically investigating the effects of epigenetic-related metabolites on epigenetics from a metabolic perspective.Metabolomics can evaluate the possibility of various modifications and the activity of modified enzymes from the perspective of the substrate and cofactor.Therefore,the purpose of this experiment is to establish a systematic targeted metabolomics method based on high performance liquid chromatography-tandem mass spectrometry to determine epigenetic metabolomes in biological samples.METHODS: Based on epigenetic modification and related enzymes,we sorted out the metabolites related to DNA and histone methylation,demethylation,histone acetylation(and other acylations),deacetylation,O-linked glycosylation,a total of 34,five types of metabolites.According to the structures and polarity of the compounds,the epigenetic related metabolites are classified into two groups: coenzyme As(CoAs)and pyridine dinucleotide and others.CoAs includes 10 acyl-CoAs such as acetyl-CoA,pyridine dinucleotides and others including NAD+(NADP+)/NADH(NADPH),S-Adenosyl methionine(SAM),S-Adenosyl-L-homocysteine(SAH),α-Ketoglutaric acid(α-KG),succinic acid,fumaric acid,O-linked glycosylation related metabolites such as N-acetylglucosamine(N-GlcNAc).Metabolites separation was performed in a liquid chromatography system using an Acquity UPLC BEH C18 column and an Acquity UPLC HSS T3 column.The ion pair reagent ammonium acetate and dibutylammonium acetate(DBAA)were introduced to the system with different ratios of water/acetonitrile as the mobile phase for gradient elution.Compounds were identified using multiple reaction monitoring modes in mass spectrometry.A standard curve corresponding to each compound was established to complete the method validation.biological samples were pretreated with methyl tert-butyl ether(MTBE)as an extraction reagent,and the measurement data was imported into MultiQuant Software 3.02 for analysis and quantification.RESULTS: The coenzyme A method established in this experiment had a quantitative range of 31.25-2000 ng,and the curve correlation coefficient was mostly greater than 0.995.The intra-day and inter-day precision were less than 20%.The accuracy was between 95% and 115%.The quantitative range of pyridine dinucleotide and other metabolites is between 62.5-2000 ng,the correlation coefficient is greater than 0.995,the inta-day and inter-day precision were less than 20%.The accuracy was between 90% and 105%.By measuring the epigenetic metabolome in the livers of aging mice with our method,we found that levels of metabolites such as methionine,S-adenosyl homocysteine,fumaric acid,UDP-GlcNAc,and FAD+ decreased.O-GlcNAc glycosylation was predicted based on changes in metabolite levels,and histone demethylation regulated by LSD1 and jmj-KDMs may reduce wit age.CONCLUSION: We established a method for quantifying epigenetic metabolome in biological samples based on high performance liquid chromatography tandem mass spectrometry and applied them in aging mouse models.The method covers most of the metabolites associated with epigenetic modification and has high sensitivity and high precision,and is suitable for systematic research on epigenetic metabolomes.By studying the mouse aging model,some differential metabolites were discovered and we can inferred some possible changes in the apparent modification pattern.