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TOWARDS HIGH-THROUGHPUT ANALYSIS OF RNA USING MASS SPECTROMETRY

MENG, ZHAOJING
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1098054876

Abstract Details

2004, PhD, University of Cincinnati, Arts and Sciences : Chemistry.
Mass spectrometry (MS) offers a number of advantages for the characterization of nucleic acids arising from its ability to provide mass and sequence information. The focus of this work is on developing mass spectrometric approaches for rapidly characterizing and quantifying ribonucleic acids (RNAs). A method using 18O labeling and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) for characterizing intact RNAs and their posttranscriptional modifications has been developed. An 18O label is incorporated at the 3’-phosphate end of oligonucleotides upon enzymatic digestion of RNAs. The combination of ESI-FTICRMS and 18O labeling facilitates identification of digestion products and possible sites of posttranscriptional modification to RNAs. Additionally, the presence of the 18O label simplifies sequence interpretation and assignment of sequence specific oligonucleotide fragment ions. The 18O labeling strategy is then used with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the relative quantitation of small RNAs. Two RNA samples were digested in normal and 18O labeled water separately and then combined for analysis. Relative ion abundances of the light- and heavy-water digestion products reveal relative quantitation information from the two RNA samples. This method should prove useful for quantitatively characterizing variations in RNA production and variations in the amount of posttranscriptionally modified nucleosides. In addition to these method developments, the use of nozzle-skimmer (NS) dissociation with sequence interpretation using existing software was investigated. Although shotgun sequencing by NS dissociation is quick and easy to implement, the approach is limited to the characterization of simple mixtures of oligonucleotides due to the complexity of the fragmentation spectrum as well as the possible overlapping of fragments from different oligonucleotides. Microfabricated devices potentially can serve as a sample preparation platform for high-throughput RNA analysis where all sample manipulations can be done on-line, faster, with less sample consumption and with the potential to be multiplexed. The background chemical noise, solvent compatibility, detection limits and ease of use of polymer-based microfabricated devices when coupled to ESI-FTICRMS were determined. It was found that such devices will be suitable for use in high-throughput analyses of RNAs.
Dr. Patrick Limbach (Advisor)
113 p.
Chemistry, Analytical
Mass Spectrometry; RNA; Method development

Recommended Citations

Citations

  • MENG, Z. (2004). TOWARDS HIGH-THROUGHPUT ANALYSIS OF RNA USING MASS SPECTROMETRY [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1098054876

    APA Style (7th edition)

  • MENG, ZHAOJING. TOWARDS HIGH-THROUGHPUT ANALYSIS OF RNA USING MASS SPECTROMETRY. 2004. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1098054876.

    MLA Style (8th edition)

  • MENG, ZHAOJING. "TOWARDS HIGH-THROUGHPUT ANALYSIS OF RNA USING MASS SPECTROMETRY." Doctoral dissertation, University of Cincinnati, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1098054876

    Chicago Manual of Style (17th edition)

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© 2004, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.