Journal of the American Chemical Society Table 1. Calculated and Observed Monoisotopic Molecular Weights of Oligonucleotides and Intermediates Present in the AlkB Reactions MW (calculated) of neutral species m/z (calculated) −4 charge monoisotopic peak m/z (observed) −4 charge monoisotopic peak lesion or base A m1A eA eA epoxide eA glycol EA EA+2 EA+16 EA+32 m6A HO-m6A 4888.86 4902.88 4912.86 4928.86 4946.87 4914.88 4916.86 4930.87 4946.87 4902.88 4918.87 1221.21 1224.71 1227.21 1231.21 1235.71 1227.71 1228.21 1231.71 1235.71 1224.71 1228.71 1221.13/16/24 1224.67 1227.22 1231.22 1235.72 1227.67 1228.16 1231.66 1235.67 1224.73 1228.72 Article (Figure 2c). In the presence of AlkB, the eA lesion was mostly converted to the undamaged product, adenine (m/z = 1221.24, Figure 2d). We also observed peak clusters consistent in MW with epoxide (1231.22) and glycol (1235.72) intermediates (Figure 1d and Figure 2d), which are consistent with previous observations.10 AlkB Repairs EA Completely. In our in vitro experiment with EA, the oligonucleotide with EA in the absence of AlkB was observed at m/z of 1227.67 for its −4 charge monoisotopic peak, which agrees well with the calculated m/z 1227.71 (Figure 3a and Table 1). In the presence of AlkB, the peak Figure 2. ESI-TOF mass spectra from reactions of alkyl adducts and AlkB protein. Data represent the −4 charge envelopes and the monoisotopic peak (all 12C, 14N, etc.) value is labeled above each peak envelope. (a) m1A, (b) m1A + AlkB, (c) eA, and (d) eA + AlkB. m/z of 1224.92, 0.25 amu larger than the 1224.67 peak, which indicates a species containing 13C, 15N, or another isotope that adds a nominal mass of 1.0 to the total weight of the 16mer (e.g., 0.25 = m/z = one 13C/charge state of 4). Additional peaks in the spectrum represent additional heavy isotopes within the parental 16mer. Model System: AlkB Repairs m1A and eA. Previously, we investigated m1A and eA lesions as substrates for AlkB both in vitro and in vivo;10,16,22 we used those two adducts as controls for the present study on the structurally similar compounds, m6A and EA. In our in vitro study of m1A repair, the lesion was completely repaired to the undamaged base adenine (Figure 2b, m/z = 1221.13), while no change occurred in the absence of AlkB (Figure 2a, m/z 1224.67). The in vitro results conﬁrm that m1A is a good substrate for AlkB, and reproduce our prior ﬁndings.16 Moreover, the current observations correlate very well with the strong reparability of m1A suggested by earlier in vivo lesion bypass studies.22 The second control compound in the present study was eA. eA was observed at m/z of 1227.22 for its −4 charge monoisotopic peak in the absence of AlkB Figure 3. ESI-TOF mass spectra from reactions of alkyl adducts and AlkB protein. Data represent the −4 charge envelopes and the monoisotopic peak (all 12C, 14N, etc.) value is labeled above each peak envelope. (a) EA, (b) EA + AlkB, (c) m6A, and (d) m6A + AlkB. envelope of EA disappeared completely after the one hour incubation. Indeed, EA was converted to four new species in the mass spectrum (Figure 3b). The peak at m/z of 1221.16 corresponds to the 16mer with an undamaged adenine base, which we did not ﬁnd in our previous study.6 One possible reason could be that the MALDI mass spectrometry tool we used earlier has poorer resolution than the current HPLC-ESI- TOF method. Another reason is that MALDI analyzes the samples immediately after the reactions, giving less time for conversion of intermediates to adenine. However, the HPLC- ESI-TOF method detects mass signals after the HPLC separation at room temperature, which might provide product and intermediate proﬁles diﬀerent than those detected in the MALDI analyses. We also controlled the HPLC autosampler temperature at 5 °C and found the formation of undamaged adenine more limited under this condition (Figure 4). Detailed analyses of the product and intermediate kinetics will be presented in a later section. We also found peaks at m/z of 1231.66 and 1235.67, which match up with intermediates with one and two extra oxygen atoms added to the EA motif, respectively. By carefully comparing the high-resolution MWs between the new species with m/z of 1228.16 and the 16mer with adenine in the lesion position, we determined that this new species has two extra atoms, consistent with the addition of one oxygen and one carbon (intermediate 6 or 9 in Figure 1a). In our previously proposed mechanism (Figure 1a, boxed), AlkB can oxidize the carbon atom attached to the N1 position 8898 dx.doi.org/10.1021/ja3010094 | J. Am. Chem. Soc. 2012, 134, 8896−8901