Fig. 3: FT-ICR mass spectrometric characterization of the methylated RNA product.
From: A natural riboswitch scaffold with self-methylation activity
a ESI mass spectrum of the 33 nt m3C Tt preQ1-I RNA; the inset shows the isotopically resolved signal of the (M–10H)10- ions; asterisks indicate piperidine RNA adducts. b Collisionally activated dissociation (CAD) of (M–nH)n– ions of RNA in the collision cell produces c and y fragment ions from RNA backbone cleavage. c Fragment-ion map illustrating sequence coverage from CAD of the methylated Tt preQ1-I RNA. The numbering of c and y fragments starts from the 5’ and 3’ terminus, respectively (top). MS signals of unmodified and methylated c13, c14, c15, c16, and complementary y20, y19, y18, y17 fragments from CAD of (M−7H)7− and (M–10H)10− ions reveal the site of methylation (C15); the calculated isotopic profiles for unmodified and singly methylated RNA are indicated by red open circles. d Loss of methylated cytosine (red) in spectra from collisionally activated dissociation (CAD) of (M–10H)10- ions of RNA is direct evidence for C nucleobase methylation; the 1:3 ratio of m3C/C nucleobase losses is consistent with destabilization of the glycosidic bond as a result of methylation at the N3 position.
