Quantification of DNA Methylation and Its Oxidized Derivatives Using LC-MS.

The amount of 5-methyl-2'-deoxycytidine (mdC) and its oxidized derivatives 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) inside mammalian cells provides valuable information concerning cellular state and fate. LC-MS methods enable reliable quantification of these noncanonical DNA modifications in the low femtomolar range. Here, we describe a broadly applicable protocol to quantify mdC, hmdC, fdC, and cadC in vertebrate-derived cells using ultra-HPLC triple quadrupole MS (UHPLC-QQQ-MS).