Identifying modifications in RNA by MALDI mass spectrometry.
Posttranscriptional modifications on the base or sugar of ribonucleosides generally result in mass increases that can be measured by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a direct and accurate means of determining the masses of RNAs. Mass spectra produced by MALDI are relatively straightforward to interpret, because they are dominated by singly charged ions, making it possible to analyze complex mixtures of RNA oligonucleotides ranging from trinucleotides up to 20-mers. Analysis of modifications within much longer RNAs, such as ribosomal RNAs, can be achieved by digesting the RNA with nucleotide-specific enzymes. In some cases, it may be desirable to isolate specific sequence regions before MALDI-MS analysis, and this requires a few additional steps. The method is applicable to the study of modified RNAs from cell extracts as well as RNA modifications added in cell-free in vitro systems. MALDI-MS is particularly useful in cases in which other techniques such as those involving primer extension or chromatographic analyses are not practicable. To date, MALDI-MS has been used to localize rRNA modifications that are involved in fundamental processes in protein synthesis as well as methylations that confer resistance to antibiotics. For several rRNA sites, MALDI-MS has served an essential role in the identification of the enzymes that catalyze the modifications.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
Stephen
S
Douthwaite
Finn
F
Kirpekar
D000818:Animals; D006801:Humans; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D012313:RNA