Mellie June Paulines, Patrick A. Limbach, Paulines, Mellie June, Limbach, Patrick A.
Editors
Alexandra Lusser
Abstract
We describe the comparative analysis of ribonucleic acid digests (CARD) approach for RNA modification analysis. This approach employs isotope labeling during RNase digestion, which allows the direct comparison of a tRNA of unknown modification status against a reference tRNA, whose sequence or modification status is known. The reference sample is labeled with (18)O during RNase digestion while the candidate (unknown) sample is labeled with (16)O. These RNase digestion products are combined and analyzed by mass spectrometry. Identical RNase digestion products will appear in the mass spectrum as characteristic doublets, separated by 2 Da due to the (16)O/(18)O mass difference. Singlets arise in the mass spectrum when the sequence or modification status of a particular RNase digestion product from the reference is not matched in the candidate (unknown) sample. This CARD approach for RNA modification analysis simplifies the determination of differences between reference and candidate samples, providing a route for higher throughput screening of samples for modification profiles, including determination of tRNA methylation patterns.
