Characterization and quantification of RNA post-transcriptional modifications using stable isotope labeling of RNA in conjunction with mass spectrometry analysis.
ABSTRACT Mass spectrometry has emerged as an increasingly powerful tool for the identification and characterization of nucleic acids, in particular RNA post-transcriptional modifications. High mass accuracy instrumentation is often required to discriminate between compositional isomers of oligonucleotides. We have used stable isotope labeling ((15)N) of E. coli RNA in conjunction with mass spectrometry analysis of the combined heavy- and light-labeled RNA for the identification and quantification of oligoribonucleotides and post-transcriptional modifications. The number of nitrogen atoms in the oligoribonucleotide and fragment ions can readily be determined using this approach, enabling the discrimination between potential compositional isomers without the requirement of high mass accuracy mass spectrometers. In addition, the identification of specific fragment ions in both the unlabeled and labeled oligoribonucleotides can be used to gain further confidence in the assignment of RNA post-transcriptional modifications. Using this approach we have identified a range of post-transcriptional modifications of E. coli 16S rRNA. Furthermore, this method facilitates the rapid and accurate quantification of oligoribonucleotides, including cyclic phosphate intermediates and missed cleavages often generated from RNase digestions.