Simultaneous determination of global DNA methylation and hydroxymethylation levels by hydrophilic interaction liquid chromatography-tandem mass spectrometry.
Methylation of DNA at the 5-position of cytosine (Cyt) is a well-studied epigenetic pathway implicated in gene silencing and embryogenesis. Recently, in addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) have been detected in certain mammalian tissues. Here, we developed and validated a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of Cyt, 5mC, and 5hmC levels in biological samples. DNA was extracted with phenol-chloroform, hydrolyzed using 88% formic acid at 140 °C, separated using a bridged ethylene hybrid HILIC column, and analyzed by tandem MS. The linearity was established over the concentration range of 1 to 500 ng/mL for Cyt, 0.2 to 100 ng/mL for 5mC, and 0.1 to 50 ng/mL for 5hmC, and the correlation coefficients were all >0.99. Limits of detection were 1 pg/mL for Cyt, 45 pg/mL for 5mC, and 57 pg/mL for 5hmC, and the limit of quantification values for Cyt, 5mC, and 5hmC were 2 pg/mL, 90 pg/mL, and 100 pg/mL, respectively. The relative standard deviation (RSD) of the intraday precision ranged from 1.87% to 4.84% and the interday precision from 2.69% to 4.98%. The recovery of the method varied from 88.25% to 104.39%. The method was then applied to the analysis of DNA from biological samples, establishing its potential for helping researchers understand the roles of modified nucleobases in DNA.
Guangdong Medical College, Zhanjiang, Guangdong, People's Republic of China.
Liangtao
L
Zhang
Lijian
L
Zhang
Keyuan
K
Zhou
Xiaoxia
X
Ye
Junjie
J
Zhang
Aimei
A
Xie
Liyu
L
Chen
Jing X
JX
Kang
Chun
C
Cai
D044503:5-Methylcytosine / Q000032:analysis / Q000031:analogs & derivatives; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D003596:Cytosine / Q000031:analogs & derivatives / Q000032:analysis; D004247:DNA / Q000032:analysis*; D019175:DNA Methylation; D057927:Hydrophobic and Hydrophilic Interactions; D057230:Limit of Detection; D053719:Tandem Mass Spectrometry / Q000379:methods*
C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA