Quantitation of Ribonucleic Acids Using 18O Labeling and Mass Spectrometry

Abstract

A previous limitation in the analysis of ribonucleic acids (RNAs) by mass spectrometry (MS) has been the inability to obtain quantitative information relating to total RNA, RNA subunits, and undermodified nucleosides in a straightforward manner. Here, a simple and rapid method has been developed for the relative quantitation of small RNAs using ¹⁸O labeling and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). One RNA sample is digested with RNase T1 in ¹⁸O-labeled (“heavy”) water with the ¹⁸O being incorporated at the 3‘-phosphate end of oligonucleotides upon hydrolysis. A second RNA sample is digested with RNase T1 in normal (“light”) water. The two samples are then combined and analyzed by MALDI-MS. Relative ion abundances of the light- and heavy-water digestion products, which are separated by 2 Da due to the isotopic mass of ¹⁸O, reveal relative quantitation information from the two RNA samples. The accuracy and reproducibility of this approach were tested on 18 known RNA samples and 4 unknown RNA samples. The coefficients of variation for quantitation were found to be generally below 15% when using MALDI-MS. The approach yields accurate quantitative information for heavy-to-light ratios greater than 1:2. This method should prove useful for quantitatively characterizing variations in RNA production and variations in the amount of posttranscriptionally modified nucleosides.