Ification of the prominent masses in these 5 peaks by operating the mass spectrometer (MS) in scanning mode, the respective precursor masses were subjected to collision induced fragmentation (CID) and the MS set to product ion scan mode. Figure 2C shows an exemplary fragmentation of a BMB-conjugate thus Title Loaded From File Title Loaded From File identified as uridine-BMB (U-BMB). An overview over all found BMB-conjugates and their fragmentation patterns used in the subsequently applied multiple reaction monitoring (MRM) method can be found in Table S1 in File S1. With this method the major peaks from the green UV chromatogram of Figure 2D were identified as -BMB (Rt=14.3 and 14.7), G-BMB (Rt=13.5 and 16.0), U-BMB (Rt=15.5 min) and s4U-BMB (Rt=16.2 min) (Figure 2E, blue chromatogram). Eluting right after U-BMB is 4hydroxylmethyl-7-methoxy-coumarin(Rt=15.9 min) which is not shown in the MS/MS chromatogram and was not further examined as it is considered a side product that does not further interfere with the labeling reaction.Considerations for semi-quantitative analysisThere are three normalization caveats in the quantitative assessment of peak integrals from LC-MS/MS runs, for each of which a normalization factor was determined. These concern (i) the injected amount of sample for which the normalization factor nA relates each peak area to that of the adenosine peak as internal standard. This normalization is displayed for BMB on the left side of Figure 3A. Here, the conjugate with uridine and guanosine (G) seem to be the most prominent reaction products. Furthermore (ii) differential detection efficiency by mass spectrometry is accounted for by the response factor rf and displayed in the middle. Now, U-BMB appears as theSpecific Alkylation of Modified NucleosidesFigure 3. Data analysis of LC-MS/MS experiments of total tRNA E. coli treated with bromomethylcoumarins. A) LC-MS/MS results from the chromatogram in Figure 2E after peak integration and data processing. The influence of the raw data processing steps is demonstrated for BMB conjugates. (i) Normalization to adenosine (nA) for intersample comparability reveals guanosine and uridine as the main reaction partners of BMB (ii). Usage of the found response factors (rf) which account for the differential ionization efficiencies in the mass spectrometer indicates U-BMB has the main reaction product. Data processing using nA and rf are used to display the reactivity of BMB (iii). Normalization to the relative abundance of tRNA modifications with the correction factor cra. This last data processing step is used to assess the selectivity of BMB for the substrate nucleosides. Here, 4-thiouridine is the main reaction partner with BMB.B) Reaction of BMB with total tRNA E. coli in comparison to 5 coumarins with different substitution patterns. On the top the chemical structure of all used coumarins are shown. The diagram below shows LC-MS/MS results of all coumarin-nucleoside conjugates from total tRNA E. coli reaction digests under slightly acidic conditions 1 (pH 6.5) monitored by their respective mass transitions (see Table S1-S6 in File S1) using the normalization to adenosine (factor nA). For comparison the 23977191 mass integrals were corrected by usage of a response factor (rf) derived from absorption at 320 nm. The colors of the bars fit to the colors of the coumarin structures above. The graph displays the results for the reaction under more alkaline conditions 2 (pH 8.25), using the same data processing. Under these conditions the reac.Ification of the prominent masses in these 5 peaks by operating the mass spectrometer (MS) in scanning mode, the respective precursor masses were subjected to collision induced fragmentation (CID) and the MS set to product ion scan mode. Figure 2C shows an exemplary fragmentation of a BMB-conjugate thus identified as uridine-BMB (U-BMB). An overview over all found BMB-conjugates and their fragmentation patterns used in the subsequently applied multiple reaction monitoring (MRM) method can be found in Table S1 in File S1. With this method the major peaks from the green UV chromatogram of Figure 2D were identified as -BMB (Rt=14.3 and 14.7), G-BMB (Rt=13.5 and 16.0), U-BMB (Rt=15.5 min) and s4U-BMB (Rt=16.2 min) (Figure 2E, blue chromatogram). Eluting right after U-BMB is 4hydroxylmethyl-7-methoxy-coumarin(Rt=15.9 min) which is not shown in the MS/MS chromatogram and was not further examined as it is considered a side product that does not further interfere with the labeling reaction.Considerations for semi-quantitative analysisThere are three normalization caveats in the quantitative assessment of peak integrals from LC-MS/MS runs, for each of which a normalization factor was determined. These concern (i) the injected amount of sample for which the normalization factor nA relates each peak area to that of the adenosine peak as internal standard. This normalization is displayed for BMB on the left side of Figure 3A. Here, the conjugate with uridine and guanosine (G) seem to be the most prominent reaction products. Furthermore (ii) differential detection efficiency by mass spectrometry is accounted for by the response factor rf and displayed in the middle. Now, U-BMB appears as theSpecific Alkylation of Modified NucleosidesFigure 3. Data analysis of LC-MS/MS experiments of total tRNA E. coli treated with bromomethylcoumarins. A) LC-MS/MS results from the chromatogram in Figure 2E after peak integration and data processing. The influence of the raw data processing steps is demonstrated for BMB conjugates. (i) Normalization to adenosine (nA) for intersample comparability reveals guanosine and uridine as the main reaction partners of BMB (ii). Usage of the found response factors (rf) which account for the differential ionization efficiencies in the mass spectrometer indicates U-BMB has the main reaction product. Data processing using nA and rf are used to display the reactivity of BMB (iii). Normalization to the relative abundance of tRNA modifications with the correction factor cra. This last data processing step is used to assess the selectivity of BMB for the substrate nucleosides. Here, 4-thiouridine is the main reaction partner with BMB.B) Reaction of BMB with total tRNA E. coli in comparison to 5 coumarins with different substitution patterns. On the top the chemical structure of all used coumarins are shown. The diagram below shows LC-MS/MS results of all coumarin-nucleoside conjugates from total tRNA E. coli reaction digests under slightly acidic conditions 1 (pH 6.5) monitored by their respective mass transitions (see Table S1-S6 in File S1) using the normalization to adenosine (factor nA). For comparison the 23977191 mass integrals were corrected by usage of a response factor (rf) derived from absorption at 320 nm. The colors of the bars fit to the colors of the coumarin structures above. The graph displays the results for the reaction under more alkaline conditions 2 (pH 8.25), using the same data processing. Under these conditions the reac.
