Metabolic isotopic labeling of (deoxy)ribose enables dual neutral loss scanning for profiling nucleic acid modifications

  • Nucleic Acids Res. 2026 Feb 24;54(5):gkag155. doi: 10.1093/nar/gkag155.
Shao-Qin Rong  1  2  3  4  5 ,  Zhi-Han Jin  1  5 ,  Dong-Rui Yin  1  5 ,  Jun Yao  1 ,  Guo-Liang Xu  1  2 ,  Ping Zhu  6  7 ,  Hai Gao  5 ,  Dan Zhou  8
Affiliations
  • 1. Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical Sciences, Medical College of Fudan University, Shanghai 200032, China.
  • 2. CAS Key Laboratory of Epigenetic Regulation and Intervention, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China.
  • 3. Advanced Molecular Pathology Institute of Soochow University and SANO, Suzhou 215123, China.
  • 4. Suzhou SANO Precision Medicine Ltd, SANO Medical Laboratories, Suzhou 215123, China.
  • 5. Shanghai Xuhui Central Hospital, Zhongshan-Xuhui Hospital, Fudan University, Shanghai 200237, China.
  • 6. Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong 510100, China.
  • 7. Guangdong Provincial Key Laboratory of Pathogenesis, Targeted Prevention and Treatment of Heart Disease, Guangzhou Key Laboratory of Cardiac Pathogenesis and Prevention, Guangzhou, Guangdong 510100, China.
  • 8. Center for Medical Research and Innovation, Shanghai Pudong Hospital, Institutes of Biomedical Sciences, Medical College of Fudan University, Shanghai 201399, China.
PMID: 41732913 DOI: 10.1093/nar/gkag155
Abstract

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.

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