Extended Data Fig. 3: Deuterium tracing of the methyl group in 5mC–DNA.

a, b, Tandem mass spectrometry analysis of the HPLC fractions corresponding to the minor side products generated in the CMD1 reaction and comparison with authentic 5hmC (a) and 5caC (b) standards (Fig. 1a; see the reaction mechanism proposed in Extended Data Fig. 7c for further discussion of the origins of 5hmC and 5caC). Data are representative of two independent experiments. c, Mass spectrometry detection of 5mC nucleoside in a DNA substrate methylated in vitro with M.SssI using D₃-labelled S-adenosyl-l-methionine ([methyl-D₃]-SAM). The mass of 5mC increases by three units when [methyl-D₃]-SAM was used. Data are representative of two independent experiments. d, Identification of P1 and P2 bases using the masses of molecules and fragmentation products from tandem mass spectrometry. P1 and P2 produce identical collision-induced dissociation (CID) fragments, suggesting that they are stereoisomers. The most abundant fragments generated by CID of P1 and P2 are shown. Molecular formulae were deduced from the molecular masses. As all the fragment ions of P1 and P2 that are generated from D₃-labelled 5mC are 2 D larger than those from unlabelled 5mC, the new modification is likely to occur at the methyl group; the bridging methylene linked to the pyrimidine ring seems unaltered in the CID. P1 and P2 appeared to lose three H₂O molecules (molecular mass 18.0100) consecutively in CID, indicating the presence of three hydroxyl groups in the P1 and P2 structures. Data are representative of two independent experiments.