Quantitative Mass Spectrometry of RNA Modifications
Ribosomal RNAs contain numerous modifications, such as base or ribose methylations and pseudouridine, in both bacteria and eukaryotic cells. Bacterial ribosomes contain over 50 modifications, yeast ribosomes contain over 75 modifications, and human ribosomes contain over 100 modifications. We are developing isotope labeling methods that allow the precise quantification of these modified residues in ribosomal RNAs.
Methylations can be conveniently monitored using metabolic deuteration with the labeled precursor D3-methionine, resulting in a +3 mass shift. Pseudouridylation can be monitored using D2-uracil, which gives a +1 mass shift after exchange with water during the isomerization reaction. Using these methods, we can quantify 29 of 35 modifications in E. coli ribosomal RNA.