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Rapid Characterization of Posttranscriptional Modifications in RNA Using Matrix Assisted Laser Desorption Ionization Mass Spectrometry and Matrix Assisted Laser Desorption Ionization Post Source Decay Mass Spectrometry

Berhane, Beniam T.

Abstract Details

2003, PhD, University of Cincinnati, Arts and Sciences : Chemistry.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful analytical tool for the structural characterization of biomolecules. However, nearly all proteomic or genomic methodologies that employ MALDI-MS require external sample manipulation which limits the overall throughput of analysis. In this work functional MALDI sample plates were fabricated to permit in-situ characterization of nucleic acids. Poly(methylmethacrylate) (PMMA) sample plates were fabricated by micromilling technique. The key structural feature of the microfabricated sample plate is the presence of individual cylindrical posts (360 µm x 360 µm), which serve as individual sample targets within the overall PMMA-based sample plate. Functionality is added to these microposts via the covalent attachment of enzymes. Enzymatic digestion of ribonucleic acids (RNAs) was performed in-situ with subsequent analysis by MALDI-MS. Advantages to such an approach include reduction in sample handling and reduction in the amount of sample required for analysis due to the small surface area of the microposts.The gas phase dissociation of biomolecules can be used for sequence analysis, structural characterization and modification analysis. MALDI-post-source decay (PSD) was used for the characterization and understanding of the fragmentation patterns that are observed in synthetic oligoribonucleotides. The results show that the PSD of synthetic oligoribonucleotides is dependent on the base composition. It was also observed that partial sequence can be obtained from the PSD fragment products.Coupling of PSD with on-probe digestion of intact RNAs was also investigated. The results show that this approach can be used for sequencing oligonucleotides and for screening the presence of known posttranscriptional modifications. To simplify PSD data analysis, stable isotope labeling was investigated by incorporating an 18 O-label at the 3’-phosphate of oligoribonucleotides during the enzymatic processing of intact RNAs. Upon enzymatic digestion, characteristic doublets representative of the isotopic distribution of oxygen were noted for those products that contain 3’-phosphate groups. This approach was used to readily distinguish endonuclease digestion products from incomplete digestion products and non-specific cleavage products. In addition, endonuclease digestion products containing the characteristic isotopic doublet can be selected for further characterization by PSD analysis. PSD products carrying the 3’-phosphate group will appear as doublet, thereby simplifying fragment ion assignment.
Dr. Patrick A. Limbach (Advisor)
110 p.

Recommended Citations

Citations

  • Berhane, B. T. (2003). Rapid Characterization of Posttranscriptional Modifications in RNA Using Matrix Assisted Laser Desorption Ionization Mass Spectrometry and Matrix Assisted Laser Desorption Ionization Post Source Decay Mass Spectrometry [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1052319621

    APA Style (7th edition)

  • Berhane, Beniam. Rapid Characterization of Posttranscriptional Modifications in RNA Using Matrix Assisted Laser Desorption Ionization Mass Spectrometry and Matrix Assisted Laser Desorption Ionization Post Source Decay Mass Spectrometry. 2003. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1052319621.

    MLA Style (8th edition)

  • Berhane, Beniam. "Rapid Characterization of Posttranscriptional Modifications in RNA Using Matrix Assisted Laser Desorption Ionization Mass Spectrometry and Matrix Assisted Laser Desorption Ionization Post Source Decay Mass Spectrometry." Doctoral dissertation, University of Cincinnati, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1052319621

    Chicago Manual of Style (17th edition)