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

Authors :: Traube FR
Schiffers S
Carell T
Source :: Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2021; Vol. 2272, pp. 77-94.
Publication Year :: 2021
Abstract: The amount of 5-methyl-2'-deoxycytidine (m <superscript>5</superscript> dC) and its oxidized derivatives 5-hydroxymethyl-dC (hm <superscript>5</superscript> dC), 5-formyl-dC (f <superscript>5</superscript> dC), and 5-carboxy-dC (ca <superscript>5</superscript> dC) 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 m <superscript>5</superscript> dC, hm <superscript>5</superscript> dC, f <superscript>5</superscript> dC, and ca <superscript>5</superscript> dC in vertebrate-derived cells using ultra-HPLC triple quadrupole MS (UHPLC-QQQ-MS).