Comparative Analysis of Ribonucleic Acid Digests (CARD) by Mass Spectrometry

  • Mellie June Paulines
  • Patrick A. LimbachEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)


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 18O during RNase digestion while the candidate (unknown) sample is labeled with 16O. 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 16O/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.

Key words

Modified nucleosides RNA sequencing tRNA rRNA Modified bases Tandem mass spectrometry LC-MS/MS MALDI-MS/MS Isotope labeling Epitranscriptome 



Financial support of this work was provided by the National Science Foundation (CHE1507357) and the University of Cincinnati.


  1. 1.
    Hopper A (2013) Transfer RNA post-transcriptional processing, turnover, and subcellular dynamics in the yeast. Genetics 194:43–67Google Scholar
  2. 2.
    Agris P (2015) The importance of being modified: an unrealized code to RNA structure and function. RNA 21:552–554CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Cantara W, Murphy F 4th, Demirci H, Agris P (2013) Expanded use of sense codons is regulated by modified cytidines in tRNA. Proc Natl Acad Sci U S A 110:10964–10969CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Weixlbaumer A, Murphy F 4th, Dziergowska A, Malkiewicz A, Vendeix F, Agris P, Ramakrishnan V (2007) Mechanism for expanding the decoding capacity of transfer RNAs by modifications of uridines. Nat Struct Mol Biol 14:498–502CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Murphy F 4th, Ramakrishnan V, Malkiewicz A, Agris P (2004) The role of modifications in codon discrimination by tRNA(Lys)UUU. Nat Struct Mol Biol 11:1186–1191CrossRefPubMedGoogle Scholar
  6. 6.
    Helm M, Brulé H, Degoul F, Cepanec C, Leroux J, Giegé R, Florentz C (1998) The presence of modified nucleotide is required for the cloverleaf folding of a human mitochondrial. Nucleic Acids Res 26:1636–1643CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pang Y, Abo R, Levine S, Dedon P (2014) Diverse cell stresses induce unique patterns of tRNA up- and down-regulation: tRNA-seq for quantifying changes in tRNA copy number. Nucleic Acids Res 42:e170CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Dedon PC, Begley TJ (2014) A System of RNA Modifications and Biased Codon Use Controls Cellular Stress Response at the Level of Translation. Chem Res Toxicol 27:330–337CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Ofengand J, Del Campo M, Kaya Y (2001) Mapping pseudouridine in RNA molecules. Methods 25:365–373CrossRefPubMedGoogle Scholar
  10. 10.
    Motorin Y, Muller S, Behm-Ansmant I, Branlant C (2007) Identification of modified residues in RNA by reverse transcription-based methods. Methods Enzymol 425:21–53CrossRefPubMedGoogle Scholar
  11. 11.
    Benedum-Wohlgamuth J, Rubio M, Paris Z (2009) Thiolation controls cytoplasmic tRNA stability and acts as a negative determinant for tRNA editing in mitochondria. J Biol Chem 284:23947–23953CrossRefGoogle Scholar
  12. 12.
    Meyer K, Saletore Y, Zumbo P, Elemento O, Mason C, Jaffrey S (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149:1635–1646CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kowalak JA, Pomerantz SC, Crain PF, McCloskey JA (1993) A novel method for the determination of post-transcriptional modification in RNA by mass spectrometry. Nucleic Acids Res 21:4577–4585CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kowalak J, Bruenger E, McCloskey J (1995) Posttranscriptional modification of the central loop of domain V in Escherichiacoli 23 S ribosomal RNA. J Biol Chem 270:17758–17764CrossRefPubMedGoogle Scholar
  15. 15.
    Wetzel C, Limbach P (2012) Global identification of transfer RNAs by liquid chromatography-mass spectrometry (LC-MS). J Proteomics 75:3450–3464CrossRefPubMedGoogle Scholar
  16. 16.
    Hossain M, Limbach PA (2007) Mass spectrometry-based detection of transfer RNAs by their signature endonuclease digestion products. RNA 13:295–303CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Li S, Limbach PA (2012) Method for comparative analysis of ribonucleic acids using isotope labeling and mass spectrometry. Anal Chem 84:8607–8613CrossRefPubMedGoogle Scholar
  18. 18.
    Li S, Limbach PA (2013) Mass spectrometry sequencing of transfer ribonucleic acids by the comparative analysis of RNA digests (CARD) approach. Analyst 138:1386–1394CrossRefPubMedGoogle Scholar
  19. 19.
    Wetzel C, Li S, Limbach PA (2014) Metabolic De-Isotoping for Improved LC-MS Characterization of Modified RNAs. J Am Soc Mass Spectrom 25:1114–1123CrossRefPubMedGoogle Scholar
  20. 20.
    Machnicka MA, Milanowska K, Osman Oglou O, Purta E, Kurkowska M, Olchowik A, Januszewski W, Kalinowski S, Dunin-Horkawicz S, Rother KM, Helm M, Bujnicki JM, Grosjean H (2013) MODOMICS: a database of RNA modification pathways--2013 update. Nucleic Acids Res 41:D262–D267CrossRefPubMedGoogle Scholar
  21. 21.
    Jühling F, Mörl M, Hartmann RK, Sprinzl M, Stadler PF, Pütz J (2009) tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic Acids Res 37:D159–D162CrossRefPubMedGoogle Scholar
  22. 22.
    Puri P, Wetzel C, Saffert P, Gaston KW, Russell SP, Varela JAC, van der Vlies P, Zhang G, Limbach PA, Ignatova Z, Poolman B (2014) Systematic identification of tRNAome and its dynamics in Lactococcus lactis. Mol Microbiol 93:944–956CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Chan P, Lowe T (2009) GtRNAdb: A database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res 37:D93–D97CrossRefPubMedGoogle Scholar
  24. 24.
    Li S, Limbach PA (2015) Identification of RNA sequence isomer by isotope labeling and LC–MS/MS. J Mass Spectrom 49:1191–1198CrossRefGoogle Scholar
  25. 25.
    Constantopoulos T, Jackson G, Enke C (1999) Effects of salt concentration on analyte response using electrospray ionization mass spectrometry. J Am Soc Mass Spectrom 10:625–634CrossRefPubMedGoogle Scholar
  26. 26.
    Sambrook J, Fritch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  27. 27.
    Pluskal T, Castillo S, Villar-Briones A, Oresic M (2010) MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics 11:395CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Rieveschl Laboratories for Mass Spectrometry, Department of ChemistryUniversity of CincinnatiCincinnatiUSA

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