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

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Book cover RNA Methylation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1562))

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 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.

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Acknowledgments

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

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Authors and Affiliations

  1. Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, 210172, Cincinnati, OH, 45221, USA

    Mellie June Paulines & Patrick A. Limbach

Authors
  1. Mellie June Paulines
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  2. Patrick A. Limbach
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Corresponding author

Correspondence to Patrick A. Limbach .

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Editors and Affiliations

  1. Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

    Alexandra Lusser

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Paulines, M.J., Limbach, P.A. (2017). Comparative Analysis of Ribonucleic Acid Digests (CARD) by Mass Spectrometry. In: Lusser, A. (eds) RNA Methylation. Methods in Molecular Biology, vol 1562. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6807-7_2

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  • DOI: https://doi.org/10.1007/978-1-4939-6807-7_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6805-3

  • Online ISBN: 978-1-4939-6807-7

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