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

The amount of 5-methyl-2'-deoxycytidine (m⁵dC) and its oxidized derivatives 5-hydroxymethyl-dC (hm⁵dC), 5-formyl-dC (f⁵dC), and 5-carboxy-dC (ca⁵dC) 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 m⁵dC, hm⁵dC, f⁵dC, and ca⁵dC in vertebrate-derived cells using ultra-HPLC triple quadrupole MS (UHPLC-QQQ-MS).