Poster abstracts

Poster number 29 submitted by Manasses Jora

Detection of ribonucleoside modifications by liquid chromatography higher-energy collisional dissociation mass spectrometry (LC-HCD-MS) and spectral matching

Manasses Jora (Department of Chemistry, University of Cincinnati), Peter A Lobue (Department of Chemistry, University of Cincinnati), Ningxi Yu (Department of Chemistry, University of Cincinnati), Robert L Ross (Department of Chemistry, University of Cincinnati), Balasubrahmanyam Addepalli (Department of Chemistry, University of Cincinnati), Patrick A Limbach (Department of Chemistry, University of Cincinnati)

Abstract:
The goal of this work is to evaluate the feasibility of higher-energy collisional dissociation (HCD)-based fragment ion fingerprints for detection of modified ribonucleosides through spectral matching. To date, more than 160 ribonucleoside modifications have been identified in different organisms [1]. The most well established approach for detection of modified nucleosides is liquid chromatography-tandem mass spectrometry (LC-MS/MS), where retention time, mass-to-charge (m/z) values of precursor and fragment ions are monitored for the observed nucleoside signal. Data processing is generally done manually, and this process is laborious, time consuming, and error prone, thus requiring well trained analysts.
Our studies show that the HCD-based dissociation of ribonucleosides at higher collisional energies (e.g., HCD 80) can create nucleoside-specific fragment ion spectra (fingerprints) resembling pseudo-MSn situation [2]. Such fingerprints have high reproducibility (intra- and interday), and are insensitive to chromatographic conditions, precursor ion abundance and sample matrix. They also enable differentiation of positional isomers exclusively through mass spectrometric behavior (i.e., precursor ion m/z and HCD-based fingerprint). In the current study, we tested the suitability of HCD fingerprints to use as LC-independent reference spectra lists for in-silico analysis of ribonucleoside modifications in a given sample. We present the observations of these studies involving the detection of modified ribonucleosides from E. coli and S. cerevisiae tRNA through NIST-MS Search-based [3] spectral matching.

References:
[1] Boccaletto, P.; Machnicka, M.A.; Purta, E.; Piatkowski, P.; Baginski, B.; Wirecki, T.W.; de Crecy-Lagard, V.; Ross, R.; Limbach, P.A.; Kotter, A.; Helm, M.; Bujnicki, J.M.. Nucleic Acids Res. 2017, 46, D303.
[2] Jora, M.; Burns, A.P.; Ross, R.L.; Lobue, P.A.; Zhao, R.; Palumbo, C.M.; Beal, P.A.; Addepalli, B.; Limbach, P.A.. J. Am. Soc. Mass Spectrom. 2018, 29, 1745.
[3] Yang, X.; Neta, P.; Stein, S.E.. J. Am. Soc. Mass Spectrom. 2017, 28, 2280.

Keywords: Ribonucleoside Modifications, LC-MSMS, Spectral Matching