Advertisement

Metabolic De-Isotoping for Improved LC-MS Characterization of Modified RNAs

  • Collin Wetzel
  • Siwei Li
  • Patrick A. LimbachEmail author
Focus: Mass Spectrometry and DNA Damage: Research Article

Abstract

Mapping, sequencing, and quantifying individual noncoding ribonucleic acids (ncRNAs), including post-transcriptionally modified nucleosides, by mass spectrometry is a challenge that often requires rigorous sample preparation prior to analysis. Previously, we have described a simplified method for the comparative analysis of RNA digests (CARD) that is applicable to relatively complex mixtures of ncRNAs. In the CARD approach for transfer RNA (tRNA) analysis, two complete sets of digestion products from total tRNA are compared using the enzymatic incorporation of 16O/18O isotopic labels. This approach allows one to rapidly screen total tRNAs from gene deletion mutants or comparatively sequence total tRNA from two related bacterial organisms. However, data analysis can be challenging because of convoluted mass spectra arising from the natural 13C and 15 N isotopes present in the ribonuclease-digested tRNA samples. Here, we demonstrate that culturing in 12C-enriched/13C-depleted media significantly reduces the isotope patterns that must be interpreted during the CARD experiment. Improvements in data quality yield a 35 % improvement in detection of tRNA digestion products that can be uniquely assigned to particular tRNAs. These mass spectral improvements lead to a significant reduction in data processing attributable to the ease of spectral identification of labeled digestion products and will enable improvements in the relative quantification of modified RNAs by the 16O/18O differential labeling approach.

Key words

Post-transcriptional modifications tRNA ncRNA Comparative analysis RNA sequencing RNase mass mapping Liquid chromatography-mass spectrometry Quantitative analysis 

Notes

Acknowledgments

Financial support of this work was provided by the National Science Foundation (CHE1212625) and a University of Cincinnati Department of Chemistry Doctoral Enhancement Award to C.W.

Supplementary material

13361_2014_889_MOESM1_ESM.pdf (137 kb)
ESM 1 (PDF 137 kb)

References

  1. 1.
    Phizicky, E.M., Hopper, A.K.: tRNA biology charges to the front. Genes Dev. 24, 1832–1860 (2010)CrossRefGoogle Scholar
  2. 2.
    Novoa, E.M., Ribas de Pouplana, L.: Speeding with control: codon usage, tRNAs, and ribosomes. Trends Genet. 28, 574–581 (2012)CrossRefGoogle Scholar
  3. 3.
    Gollnick, P., Babitzke, P.: Transcription attenuation. Biochem. Biophys. Acta 1577, 240–250 (2002)Google Scholar
  4. 4.
    Dittmar, K.A., Mobley, E.M., Radek, A.J., Pan, T.: Exploring the regulation of tRNA distribution on the genomic scale. J. Mol. Biol. 337, 31–47 (2004)CrossRefGoogle Scholar
  5. 5.
    Wohlgemuth, S.E., Gorochowski, T.E., Robous, J.A.: Translational sensitivity of the Escherichia coli genome to fluctuating tRNA availability. Nucleic Acids Res. 41, 8021–8033 (2013)CrossRefGoogle Scholar
  6. 6.
    Hershberg, R., Petrov, D.A.: Selection on codon bias. Annu. Rev. Genet. 42, 287–299 (2008)CrossRefGoogle Scholar
  7. 7.
    Cantara, W.A., Crain, P.F., Rozenski, J., McCloskey, J.A., Harris, K.A., Zhang, X., Vendeix, F.A.P., Fabris, D., Agris, P.F.: The RNA modification database, RNAMDB: 2011 update. Nucleic Acids Res. 39, D195–D201 (2011)CrossRefGoogle Scholar
  8. 8.
    Machnicka, M., Milanowska, K., Osman, O., Purta, E., Kurkowska, M., Olchowik, A., Januszewski, W., Kalinowski, S., Dunin-Horkawicz, S., Rother, K., Helm, M., Bujnicki, J., Grosjean, H. MODOMICS: a database of RNA modification pathways—2013 update. Nucleic Acids Res. D262–267 (2012)Google Scholar
  9. 9.
    Graeber, M.B., Muller, U.: Recent developments in the molecular genetics of mitochondrial disorders. J. Neurol. Sci. 153, 251–263 (1998)CrossRefGoogle Scholar
  10. 10.
    Saikia, M., Fu, Y., Pavon-Eternod, M., He, C., Pan, T.: Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs. RNA 16, 1317–1327 (2010)CrossRefGoogle Scholar
  11. 11.
    Maynard, N.D., Macklin, D.N., Kirkegaard, K., Covert, M.W.: Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting. Mol. Syst. Biol. 8, 567 (2012)CrossRefGoogle Scholar
  12. 12.
    Florentz, C.: Molecular investigations on tRNAs involved in human mitochondrial disorders. Biosci. Rep. 22, 81–98 (2002)CrossRefGoogle Scholar
  13. 13.
    Gebetsberger, J., Zywicki, M., Kunzi, A., Polacek, N.: tRNA-derived fragments target the ribosome and function as regulatory non-coding RNA in Haloferax volcanii. Archaea 2012, 11 (2012)CrossRefGoogle Scholar
  14. 14.
    Gingold, H., Dahan, O., Pilpel, Y.: Dynamic changes in translational efficiency are deduced from codon usage of the transcriptome. Nucleic Acids Res. 40, 10053–10063 (2012)CrossRefGoogle Scholar
  15. 15.
    Chang, D.-E., Smalley, D., Conway, T.: Gene expression profiling of Escherichia coli growth transitions: an expanded stringent response model. Mol. Microbiol. 45, 289–306 (2003)CrossRefGoogle Scholar
  16. 16.
    Moukadiri, I., Garzon, M.J., Bjork, G.R., Armengod, M.E.: The output of the tRNA modification pathways controlled by the Escherichia coli MnmEG and MnmC enzymes depends on the growth conditions and the tRNA species. Nucleic Acids Res. 42, 2602–2623 (2014)Google Scholar
  17. 17.
    Chan, C.T., Dyavaiah, M., DeMott, M.S., Taghizadeh, K., Dedon, P.C., Begley, T.J.: A quantitative systems approach reveals dynamic control of tRNA modifications during cellular stress. PLoS Genet. 6, e1001247 (2010)CrossRefGoogle Scholar
  18. 18.
    Dumelin, C., Chen, Y., Leconte, A., Chen, Y., Liu, D.: Discovery and biological characterization of geranylated RNA in bacteria. Nat. Chem. Biol. 8, 913–919 (2012)Google Scholar
  19. 19.
    Pavon-Eternod, M., Gomes, S., Geslain, R., Dai, Q., Rosner, M.R., Pan, T.: tRNA over-expression in breast cancer and functional consequences. Nucleic Acids Res. 37, 7268–7280 (2009)CrossRefGoogle Scholar
  20. 20.
    Dong, H., Nilsson, L., Kurland, C.G.: Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J. Mol. Biol. 260, 649–663 (1996)CrossRefGoogle Scholar
  21. 21.
    Liu C, B.T., Hou YM.: Fluorophore labeling to monitor tRNA dynamics. Methods Enzymol. 469, 69–93 (2009)Google Scholar
  22. 22.
    Yokogawa, T., Kumazawa, Y., Miura, K., Watanabe, K.: Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method. Nucleic Acids Res. 17, 2623–2638 (1989)CrossRefGoogle Scholar
  23. 23.
    Mir, K.U., Southern, E.M.: Determining the influence of structure on hybridization using oligonucleotide arrays. Nat. Biotechnol. 17, 788–792 (1999)CrossRefGoogle Scholar
  24. 24.
    McCloskey, J.A., Nishimura, S.: Modified nucleosides in transfer RNA. Acc. Chem. Res. 10, 403–409 (1977)CrossRefGoogle Scholar
  25. 25.
    Kowalak, J.A., Pomerantz, S.C., Crain, P.F., McCloskey, J.A.: A novel method for the determination of post-transcriptional modification in RNA by mass spectrometry. Nucleic Acids Res. 21, 4577–4585 (1993)CrossRefGoogle Scholar
  26. 26.
    Douthwaite, S., Kirpekar, F.: Identifying modifications in RNA by MALDI mass spectrometry. Methods Enzymol. 425, 1–20 (2007)CrossRefGoogle Scholar
  27. 27.
    Huang, T.Y., Liu, J., McLuckey, S.A.: Top-down tandem mass spectrometry of tRNA via ion trap collision-induced dissociation. J. Am. Soc. Mass Spectrom. 21, 890–898 (2010)CrossRefGoogle Scholar
  28. 28.
    Matthiesen, R., Kirpekar, F.: Identification of RNA molecules by specific enzyme digestion and mass spectrometry: software for and implementation of RNA mass mapping. Nucleic Acids Res. 37, e48 (2009)CrossRefGoogle Scholar
  29. 29.
    Suzuki, T., Ikeuchi, Y., Noma, A., Sakaguchi, Y.: Mass spectrometric identification and characterization of RNA-modifying enzymes. Methods Enzymol. 425, 211–229 (2007)CrossRefGoogle Scholar
  30. 30.
    Taucher, M., Breuker, K.: Characterization of modified RNA by top-down mass spectrometry. Angew. Chem. Int. Ed. Engl. 51, 11289–11292 (2012)CrossRefGoogle Scholar
  31. 31.
    Buvoli, A., Buvoli, M., Leinwand, L.A.: Enhanced detection of tRNA isoacceptors by combinatorial oligonucleotide hybridization. RNA 6, 912–918 (2000)CrossRefGoogle Scholar
  32. 32.
    Hossain, M., Limbach, P.A.: Mass spectrometry-based detection of transfer RNAs by their signature endonuclease digestion products. RNA 13, 295–303 (2007)CrossRefGoogle Scholar
  33. 33.
    Wetzel, C., Limbach, P.: The global identification of tRNA isoacceptors by targeted tandem mass spectrometry. Analyst 138, 6063–6072 (2013)CrossRefGoogle Scholar
  34. 34.
    Li, S., Limbach, P.: Method for Comparative Analysis of Ribonucleic Acids Using Isotope Labeling and Mass Spectrometry. Anal. Chem. 84, 8607–8613 (2012)CrossRefGoogle Scholar
  35. 35.
    Castleberry, C.M., Limbach, P.: Relative quantitation of transfer RNAs using liquid chromatography mass spectrometry and signature digestion products. Nucleic Acids Res. 38, e162 (2010)CrossRefGoogle Scholar
  36. 36.
    Wetzel, C., Limbach, P.: Global identification of transfer RNAs by liquid chromatography-mass spectrometry (LC-MS). J. Proteome 75, 3450–3464 (2011)CrossRefGoogle Scholar
  37. 37.
    Li, S., Limbach, P.: Mass spectrometry sequencing of transfer ribonucleic acids by the comparative analysis of RNA digests (CARD) approach. Analyst 138, 1386–1394 (2013)CrossRefGoogle Scholar
  38. 38.
    Meng, Z., Limbach, P.: Quantitation of Ribonucleic Acids using 18O labeling and mass spectrometry. Anal. Chem. 77, 1891–1895 (2005)CrossRefGoogle Scholar
  39. 39.
    Castleberry, C.M., Lilleness, K., Baldauff, R., Limbach, P.A.: Minimizing 18O/16O back-exchange in the relative quantification of ribonucleic acids. J. Mass Spectrom. 44, 1195–1202 (2009)CrossRefGoogle Scholar
  40. 40.
    Murakami, S., Fujishima, K., Tomita, M., Kanai, A.: Metatranscriptomic analysis of microbes in an oceanfront deep-subsurface hot spring reveals novel small RNAs and type-specific tRNA degradation. Appl. Environ. Microbiol. 78, 1015–1022 (2012)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2014

Authors and Affiliations

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

Personalised recommendations