Ultra-performance liquid chromatography.pdf

U a X a b c a A R A A K G H U 1 m b a a l v s t w h c m p c 1 dJournal of Chromatography B, 879 (2011) 1647–1652 Contents lists available at ScienceDirect Journal of Chromatography B journa l homepage: www.e lsev ier .com/ locate /chromb ltra-performance liquid chromatography/tandem mass spectrometry for ccurate quantification of global DNA methylation in human sperms iaoli Wanga, Yongshan Suoc, Ruichuan Yina, Heqing Shenb,??, Hailin Wanga,? State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China Reproduction Center of Zaozhuang Maternity and Child Care Hospital, Zaozhuang 277102, China r t i c l e i n f o rticle history: eceived 15 November 2010 ccepted 3 April 2011 vailable online 12 April 2011 eywords: lobal DNA methylation uman sperm PLC–MS/MS a b s t r a c t Aberrant DNA methylation in human sperms has been proposed to be a possible mechanism associated with male infertility. We developed an ultra-performance liquid chromatography/tandem mass spec- trometry (UPLC–MS/MS) method for rapid, sensitive, and specific detection of global DNA methylation level in human sperms. Multiple-reaction monitoring (MRM) mode was used in MS/MS detection for accurate quantification of DNAmethylation. The intra-day and inter-day precision values of this method were within 1.50–5.70%. By using 2-deoxyguanosine as an internal standard, UPLC–MS/MS method was applied for the detection of global DNA methylation levels in three cultured cell lines. DNA methyl- transferases inhibitor 5-aza-2′-deoxycytidine can significantly reduce global DNA methylation levels in treated cell lines, showing the reliability of our method. We further examined global DNA methylation levels in human sperms, and found that global methylation values varied from 3.79% to 4.65%. The