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Taking It Down a Level: Low Flow Sampling of RNAs by Liquid Chromatography Coupled to Tandem Mass Spectrometry

Ross, Robert L
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1470757778

Abstract Details

2016, PhD, University of Cincinnati, Arts and Sciences: Chemistry.
Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA), these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. These modifications can be present at very low levels and their analysis can be challenging. The goal of this dissertation was to examine new methods and technologies, which may allow for the analysis of chemical modifications from milligram amounts of initial biomass. Method developments were done using tRNA, which contains the most modifications of any RNA species. The methods described herein have allowed for tRNA nucleoside analysis on biological samples an order of magnitude less in scale than common in the field. Optimization of the extraction protocol by the use of proteinase K has allowed for systematic reproducible extraction and analysis on standard liquid chromatography coupled to tandem mass spectrometry platform. The use of new sample introduction technologies was shown to decrease the level of detection (LOD) from picomole concentrations to femtomole levels, a 50-fold reduction yielding a promising new method for sample introduction. Finally, the exploration of a new stationary phase for capillary chromatography allowed LC-MS/MS analysis on the nanogram scale. Altogether, the methods described here form a platform for further exploration of technologies that push the limit of LC-MS/MS sampling of RNA.
Patrick Limbach, Ph.D. (Committee Chair)
William Heineman, Ph.D. (Committee Member)
Edward Merino, Ph.D. (Committee Member)
92 p.
Analytical Chemistry

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Citations

  • Ross, R. L. (2016). Taking It Down a Level: Low Flow Sampling of RNAs by Liquid Chromatography Coupled to Tandem Mass Spectrometry [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1470757778

    APA Style (7th edition)

  • Ross, Robert. Taking It Down a Level: Low Flow Sampling of RNAs by Liquid Chromatography Coupled to Tandem Mass Spectrometry. 2016. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1470757778.

    MLA Style (8th edition)

  • Ross, Robert. "Taking It Down a Level: Low Flow Sampling of RNAs by Liquid Chromatography Coupled to Tandem Mass Spectrometry." Doctoral dissertation, University of Cincinnati, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1470757778

    Chicago Manual of Style (17th edition)

ucin1470757778
291
© 2016, some rights reserved.Creative Commons License Taking It Down a Level: Low Flow Sampling of RNAs by Liquid Chromatography Coupled to Tandem Mass Spectrometry by Robert L Ross is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Cincinnati and OhioLINK.