Post-transcriptional RNA modifications
that are introduced during
the multistep ribosome biogenesis process are essential for protein
synthesis. The current lack of a comprehensive method for a fast quantitative
analysis of rRNA modifications significantly limits our understanding
of how individual modification steps are coordinated during biogenesis
inside the cell. Here, an LC-MS approach has been developed and successfully
applied for quantitative monitoring of 29 out of 36 modified residues
in the 16S and 23S rRNA from Escherichia coli. An isotope labeling strategy is described for efficient identification
of ribose and base methylations, and a novel metabolic labeling approach
is presented to allow identification of MS-silent pseudouridine modifications.
The method was used to measure relative abundances of modified residues
in incomplete ribosomal subunits compared to a mature <sup>15</sup>N-labeled rRNA standard, and a number of modifications in both 16S
and 23S rRNA were present in substoichiometric amounts in the preribosomal
particles. The RNA modification levels correlate well with previously
obtained profiles for the ribosomal proteins, suggesting that RNA
is modified in a schedule comparable to the association of the ribosomal
proteins. Importantly, this study establishes an efficient workflow
for a global monitoring of ribosomal modifications that will contribute
to a better understanding of mechanisms of RNA modifications and their
impact on intracellular processes in the future