Detection of four BH-labeled normal deoxynucleotides (C: dCMP at 503.144, T: TMP at 518.144, A: dAMP at 527.155, G: dGMP at 543.151) and five BH-labeled DNA adducts (1,N⁶-etheno-dAMP at 551.156, 8-oxo-dGMP at 559.146, N²-ethyl-dGMP at 571.182, BP-A at 829.248, and BP-G at 845.245), along with an internal standard (N²-ethyl-d4-dGMP at 575.209) by MALDI-TOF-MS. A sample volume of 0.5 μL containing 50 fmol each of these compounds, that was also 0.5 mg/mL in α-cyano-4-hydroxycinnamic acid (CCA matrix) in 50% in acetonitrile, was deposited manually onto a stainless steel plate (AB SCIEX V70066), and the spectrum was summed from 50 laser pulses on a MALDI-TOF-MS (AB SCIEX Voyager DE STR) in the reflectron mode. BH, benzoylhistamine; dN (the deoxynucleoside part of a normal DNA mononucleotide); dN(X), a DNA adduct; BP-A, benzo[a]pyrene diolepoxide adduct of dAMP; BP-G, benzo[a]pyrene diolepoxide adduct of dGMP. The observed masses matched the exact masses exactly except for A (exact mass 527.156), ethyl-G-d4 (527.207), and BP-A (829.250). Internal calibration was done: the peaks for C, BP-G, and etheno-A were employed, but their masses were allowed to vary in the calculation.

Daughter ions (post-source collision-induced dissociation of M⁻ at 551.17) from BH-labeled 1,N⁶-etheno-dA. A sample volume of 40 nL containing 80 amol of this compound in 5 mg/mL CCA matrix with 0.5 mg/mL ammonium citrate in 50% acetontrile was deposited onto a stainless steel plate (AB SCIEX 1018497) using a TEMPO (AB-SCIEX), and the spectrum was summed from 400 laser pulses on a MALDI-TOF/TOF-MS (AB SCIEX 5800).

Detection of isotopologue BH-deoxynucleotides from placental DNA by negative ion MALDI-TOF-MS. A: fC-BHdnt; B: hmC-BHdnt; C: putative 6-oxo-T-BHdnt, designated here as oT. Exact masses: fC (531.1393), hmC (533.1550), oT (534.1390).

Larger m/z scales for the corresponding data shown in Figure 3. The peak pairs marked with an asteric may be artifacts, e.g. m/z 586 and 585 might be an adduct ion of BH-phosphate-sugar with matrix. G: BH-GMP.

Fragmentation analysis by negative ion MALDI-TOF-MS of deprotonated molecules of: A, fC-BHdnt; B: hmC-BHdnt; C: oT-BHdnt.

Fragmentation analysis by positive ion MALDI-TOF/TOF-MS of protonated nucleobases formed in the ion source from the following samples: A: hmC-BHdnt isolated from placental DNA; B: 5-hydroxymethyl-2′-deoxyuridine (purchased).

Positive ion MALDI-TOF-MS spectrum of thymidine after reaction with hydrogen peroxide followed by MALDI-MS. A peak for protonated thymine nucleobase (formed in the ion source) is seen at m/z 127, along with a peak at m/z 143 which corresponds to protonated putative 6-oxo-thymine (and also, in principle, protonated 5-hydroxymethyluracil).

Positive ion MALDI-TOF/TOF-MS of the sample tested in Figure 6. A. Spectrum from CID of m/z 127. B. Spectrum from CID of m/z 143. Accompanying losses of hydrogen atoms are not always depicted.