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High-Throughput De Novo Sequencing of Transfer RNAs Using Liquid Chromatography-Tandem Mass Spectrometry

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2013, MS, University of Cincinnati, Arts and Sciences: Chemistry.
Post-transcriptional modifications of ribonucleic acids (RNA) play an important role in the structural and functional activities of these biomolecules. Despite the importance of these modifications, very little is known relating to the diversity of RNA modification at the organism level. What is typically known is the identity of such modifications but not their sequence locations. For example, total transfer RNA (tRNA) sequences, including sites of modification, are known for only a few organisms. The goal of this thesis is to create a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that can be used to obtain modified sequences from total cellular tRNAs without requiring isolating each individual tRNA. Modifications on about 60 different positions throughout 47 different tRNAs in Thermus thermophilus were identified. New approaches to improve the identification of RNase digestion products from tRNAs were developed. The effects of segmented scans versus a single scan, shallow versus steep LC gradients and sample loading levels on success of modification placement were compared. Tandem mass spectrometry was used to confirm digestion product sequences. These improvements not only identified more modified sequences of total tRNAs, they also provided an overview of the effect of different analytical parameters on total tRNA analysis.
Patrick Limbach, Ph.D. (Committee Chair)
Joseph Caruso, Ph.D. (Committee Member)
Pearl Tsang, Ph.D. (Committee Member)
78 p.

Recommended Citations

Citations

  • Shi, W. (2013). High-Throughput De Novo Sequencing of Transfer RNAs Using Liquid Chromatography-Tandem Mass Spectrometry [Master's thesis, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1378197247

    APA Style (7th edition)

  • Shi, Wunan. High-Throughput De Novo Sequencing of Transfer RNAs Using Liquid Chromatography-Tandem Mass Spectrometry. 2013. University of Cincinnati, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1378197247.

    MLA Style (8th edition)

  • Shi, Wunan. "High-Throughput De Novo Sequencing of Transfer RNAs Using Liquid Chromatography-Tandem Mass Spectrometry." Master's thesis, University of Cincinnati, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1378197247

    Chicago Manual of Style (17th edition)