Poster abstracts

Poster number 51 submitted by Colette Castleberry

Quantitation of Isotopically Labeled RNA Using LC-MS

Colette M. Castleberry (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati), Patrick A. Limbach (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati)

Abstract:
The coupling of high performance liquid chromatography (HPLC) to electrospray ionization mass spectrometry (ESI-MS) has proven to be a formidable technique for the separation and analysis of oligonucleotide mixtures. The use of matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) to analyze and quantify isotopically labeled ribonucleic acids (RNAs) has provided the ability to analyze defined and known mixtures; however, the analysis and quantification of complex mixtures is still limited by this technique. Capillary LC coupled to ESI-MS is currently under development in order to separate and quantify complex mixtures of RNA. For method development, incorporation of the isotope is performed by digestion of a known RNA in the presence of H216O or H218O. Labeled oligonucleotide fragments are then separated with ion pairing chromatography using triethylamine and 1,1,1,3,3,3-hexafluoroisopropanol as ion pairing agents. Mass spectrometric analysis reveals the relative abundance of 16O and 18O labeled oligonucleotides. Although current method development has been performed with Escherichia coli (MRE 600), this method will be applicable to biological systems wherein RNA abundances vary due to growth or other environmental conditions.

Keywords: Mass Spectrometry, High Performance Liquid Chromatography