Marcaje isotópico metabólico de (desoxi)ribosa para escaneo de pérdida neutra dual en el perfilado de modificaciones...

Modifications on DNA and RNA, even at trace levels, play critical roles in diverse biological processes, yet their accurate quantification and discovery of novel modified constituents remain challenging. Here, we present a metabolic (deoxy)ribose-labeling approach integrated with multiple reaction monitoring mass spectrometry (MRM-MS) to enable sensitive and untargeted detection of nucleic acid modifications. In this approach, [U-13C]glucose is used to generate an ∼1:1 ratio of ¹³C5-labeled and unlabeled deoxyribose moieties in vivo, followed by MRM-MS acquisition of neutral-loss transitions corresponding to both isotopic forms. This isotopic pairing facilitates the differentiation of endogenous nucleosides from contaminants and allows confident assignment of authentic nucleoside signals. Applying this method to mouse embryonic stem cells, we detected rare nucleoside species such as 5-formylcytosine and 5-carboxycytosine, present at frequencies as low as one in 106-107 bases. In contrast, peaks assigned to N6-methyladenine (6mA) lacked a labeled counterpart, suggesting that previously reported 6mA in mammalian DNA may arise from RNA misincorporation or artifacts introduced during the processing of isolated DNA. Analysis of formaldehyde-treated DNA revealed several previously unreported adducts, including N4-hydroxymethyl-5-hydroxymethylcytosine (4hm5hmC, or dihmC). Collectively, this (deoxy)ribose-labeling strategy provides a robust and sensitive platform for untargeted nucleoside profiling and the discovery of uncharacterized nucleic acid modifications.