Poster abstracts

Poster number 30 submitted by Collin Wetzel

Identification and sequencing of transfer ribonucleic acid (tRNA) signature digestion products through the use of high pressure liquid chromatography (HPLC) and Ultra-high pressure Liquid chromatography (UPLC) coupled to an ion trap mass spectrometer

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

Abstract:
Mass spectrometry (MS) gives a unique approach to the problem of RNA sequencing and post transcriptional modification status. In MS analysis individual molecules can be detected and identified by their mass to charge ratio (m/z), thus a RNA can easily be identified based on a theoretical m/z value. The identity of these molecules can further be confirmed and sequenced by the use of MS/MS. Large strands of RNA like tRNA it must be enzymatically digested into smaller fragments prior to analysis. Every enzyme produces different digestion products based on where they cause cleaves. For the detection of more than one tRNA each must have a digestion product with a unique m/z value specific to that tRNA. These digestion products are known as signature digestion products.
High pressure Liquid chromatography (HPLC) is a technique that is often coupled with MS detectors to aid in the analysis of complex samples by separating the components of a mixture. A variation of this technique ultra high pressure Liquid chromatography (UPLC) which utilizes a smaller sorbent size, which has been known to cause better resolution but causes higher back pressure on the instrument. The smaller particles reduce the amount of diffusion during the separation process leading to narrower sample plugs entering the detector.
In this comparison E. coli total tRNA was digested into smaller products using Ribonuclease (RNase) T1. These digestion products were then separated using two different LC columns and analyzed on a linear ion trap mass spectrometer. One hundred and five products with signature mass values corresponding to specific tRNAs were attempted to be detected and verified by their sequencing information. Of the 105 possible signature digestion products for E.coli total tRNA after digestion with the T1 enzyme 90 were able to be identified but only 40 were able to be confirmed with their CID fragmentation with the use of UPLC separation. Only 73 products were able to be identified using HPLC and 37 of these products were able to be confirmed by their CID fragmentation.

Keywords: UPLC, Mass spectrometry