Poster abstracts

Poster number 88 submitted by Collin Wetzel

Targeted LC-MS/MS Identification of tRNA Through the Detection of Unique Transitions

Collin Wetzel (Chemistry, University of Cincinnati), Patrick Limbach (Chemistry, University of Cincinnati)

Abstract:
The global identification of individual transfer ribonucleic acids (tRNAs) can prove to be very difficult, requiring rigorous sample preparation prior to analysis due to complex tertiary structures made up of several highly conserved structural motifs. Our lab has previously shown LC-MS approaches to characterize endonuclease digestion products of tRNAs through the detection of “Signature Digestion Products” (SDPs) is an ideal way for global analysis. While this is a simple and novel approach it has limitations. The use of an MS/MS approach adds a degree of selectivity to the detection aspect increasing identification confidence, decreasing sample analysis time and increases the total number of tRNA detectable through the use of one enzymatic digestion. For example, in E.coli when using RNase T1, only 28 of the 47 tRNA isoacceptors are able to be identified by LC-MS analysis of SDPs alone; MS/MS analysis increases this number to 39. Furthermore, we have discovered only 18 precursor m/z values need to be targeted to obtain enough unique transitions to detect these 39 tRNA isoacceptors. This minimal detection requirement combined with steep LC gradients and fast linear velocities yield fast sample analysis time only 9 minutes, while minimizing sensitivity losses due to MS/MS vs. MS analysis. The extremely high linear velocity used in these experiments was made possible by the utilization of solid core RP-LC technology found in the Poroshell® column provided by Agilent.

References:
Hossain, M. & Limbach, P.A. RNA 13, 295 (2007).
Hossain, M. & Limbach, P.A. Anal. Bioanal. Chem (2008).
Castleberry, M.C. & Limbach, P.A. Nucleic Acids Res. (2010)

Keywords: Oligonucleotide, tRNA, MSMS