Figure 3. MS-based identification of acetylcytidines in S. pombe 18S rRNA.

A. Extracted ion monitoring of RNase T1 fragments of 18S rRNA containing acetylcytidine (AcC) and cytidine (C)-1297 (upper panel) and AcC and C-1815 (lower panel). The 18S rRNAs were purified from strain SP6 or Nat10_G285D grown at 30°C in YE medium, digested by RNase T1 and applied to the LC-MS system (50 fmol each). The sequences and m/z values of AcC and C-containing nucleotides are indicated. A mass window of 3 ppm was used for the extractions. Y axis indicates the peak intensity relative to the most intensive peak in each panel. Note that the MS signals of AcC-containing nucleotides were completely lost in the Nat10_G285D mutant strain (indicated by arrows). B. MS/MS spectra of AcC-containing fragments. The acetylated RNA ions [C(AcC)Gp¹⁻, m/z 1014.14; UUUC(AcC)Gp²⁻, m/z 965.60, in A) were analyzed by collision-induced dissociation. The position of acetylcytidine residues was identified by manual interpretation of the a-, c-, w- and y-type series ions and other specific product ions as indicated in the figure. The series ions assigned are indicated on the RNA sequence in the inset.