title: Comparative Analysis of Ribonucleic Acid Digests (CARD) by Mass Spectrometry.
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 with18O during RNase digestion while the candidate (unknown) sample is labeled with16O. 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 the16O/18O 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.
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, 210172, Cincinnati, OH, 45221, USA.
authors.forename: Mellie June
authors.initials: MJ
authors.lastname: Paulines
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, 210172, Cincinnati, OH, 45221, USA. Pat.Limbach@uc.edu.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
mesh_terms: D002853:Chromatography, Liquid; D044127:Epigenesis, Genetic; D020869:Gene Expression Profiling; D006868:Hydrolysis; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D059467:Transcriptome
chemical_list: D012313:RNA
keywords: Epitranscriptome; Isotope labeling; LC-MS/MS; MALDI-MS/MS; Modified bases; Modified nucleosides; RNA sequencing; Tandem mass spectrometry; rRNA; tRNA