HPLC-MS-MS method for low level DNA methylation value determination in ecological species
HU Jun-jie1, WU Yi-chong1, CHEN Gui-lian1, GAO Yuan-yuan2, ZHAO Tong3, YU Guang1, LAN Shan-hong1, Lü Xiao-mei1
1. School of Environment and Civil Engineering, Dongguan University of Technology, Dongguan 523808, China; 2. Environmental Technology Center of Dongguan, Dongguan 523009, China; 3. College of Resources and Environment, Yangtzeu University, Wuhan 434023, China
Abstract：DNA methylation patterns which associated with normal tissue development and disease initiation could be affected by environmental contaminants and related toxicity. For the requirement of quantification low abundant of 5-methyl-2’-deoxycytidine (5-mdC) in the genome of ecologically relevant species, a label-free and accurate method using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was developed and validated. The global DNA methylation calculation formula of 5-mdC(mole)/(5-mdC(mole)+dC(mole)) could be transformed as 1/(1+dC(mole)/5-mdC(mole)), then the molar ratio of 5-mdC and dC (dC(mole)/5-mdC(mole)) in DNA samples was obtained by HPLC-MS/MS. The important features of the provided method are as follows: (1) avoiding the usage of expensive stable isotope labeled internal standards; (2) isocratic separating normal and modified nucleosides and eliminating cross-interferences of analytes; (3) providing data with high accuracy even detecting low levels of methylated DNA samples. The limits of quantification for 5-mdC and dC were 40 and 60pg/mL, respectively. The intra-day and inter-day relative standard deviations (≤10%) and accuracy (relative errors) were satisfactory. The results obtained from the analysis of calf thymus DNA and genomic DNA from Daphnia magna and elegans demonstrated the feasibility of the new developed method in evaluation of the potential epigenetic risk of environmental pollutions on ecologically relevant species.
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