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Higher-energy Collisional Dissociation (HCD) as a Complementary Tool for Expanding the Detection and Discovery of Modified Ribonucleosides by LC-MS/MS

Jora, Manasses
http://orcid.org/0000-0002-1999-1508
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1613747846885246

Abstract Details

2020, PhD, University of Cincinnati, Arts and Sciences: Chemistry.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the gold-standard technique for the discovery, detection and structural characterization of post-transcriptional ribonucleoside modifications. In this technique, the modification is assigned based on the diagnostic nucleobase ion (BH2+) observed following cleavage of the N-glycosidic bond of the protonated ribonucleoside molecular ion (MH+), through a process referred to as collision-induced dissociation (CID). This nearly universal fragmentation pathway enables detection of modifications that reside on the nucleobase or on the ribose sugar. Various laboratories have exploited this feature to create powerful analytical platforms for the characterization of known and unknown modifications. Orbitrap-based mass spectrometers offer higher-energy collisional dissociation (HCD) as an alternate tool for fragmentation of molecular ion precursors. In this dissertation, I demonstrate the utility of HCD to generate nucleoside-specific MS/MS spectra (i.e., fingerprints) for the characterization of modified ribonucleosides. The quality of such fingerprint data is insensitive to chromatographic conditions, precursor ion abundance and sample matrix. The studies herein shown demonstrate the capabilities of HCD fingerprints for: (a) accurate identification of positional isomers of ribonucleosides (e.g., 3-, N4-, and 5-methylcytidine) exclusively based on their mass spectrometric features (i.e., MH+ mass-to-charge ratio – m/z, and HCD-based MS/MS fingerprint); (b) discovery and structural characterization of ribonucleosides (known, new, or artifacts) through fingerprint comparisons; (c) rapid detection of known RNA modifications with high sensitivity and specificity in a retention time-independent fashion; and (d) label-free discovery of new ribonucleosides through spectral matching. Such an accurate and rapid detection platform is expected to accelerate research on the discovery and biological significance of post-transcriptional ribonucleoside modifications.
Patrick Limbach, Ph.D. (Committee Chair)
Balasubrahmanyam Addepalli, Ph.D. (Committee Member)
George Stan, Ph.D. (Committee Member)
Ryan White, Ph.D. (Committee Member)
211 p.
Analytical Chemistry
HCD; Nucleoside; RNA modification; Spectral Network

Recommended Citations

Citations

  • Jora, M. (2020). Higher-energy Collisional Dissociation (HCD) as a Complementary Tool for Expanding the Detection and Discovery of Modified Ribonucleosides by LC-MS/MS [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1613747846885246

    APA Style (7th edition)

  • Jora, Manasses. Higher-energy Collisional Dissociation (HCD) as a Complementary Tool for Expanding the Detection and Discovery of Modified Ribonucleosides by LC-MS/MS. 2020. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1613747846885246.

    MLA Style (8th edition)

  • Jora, Manasses. "Higher-energy Collisional Dissociation (HCD) as a Complementary Tool for Expanding the Detection and Discovery of Modified Ribonucleosides by LC-MS/MS." Doctoral dissertation, University of Cincinnati, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1613747846885246

    Chicago Manual of Style (17th edition)

ucin1613747846885246
42
© 2020, some rights reserved.Creative Commons License Higher-energy Collisional Dissociation (HCD) as a Complementary Tool for Expanding the Detection and Discovery of Modified Ribonucleosides by LC-MS/MS by Manasses Jora 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.