Poster abstracts

Poster number 6 submitted by Colette Castleberry

Relative Quantitation of Isotopically Labeled RNAs Using Data Dependent LC-MS/MS

Colette M. Castleberry (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati), Rachel M. Selby (Department of Chemistry, Rose-Hulman Institute of Technology), Larry Sallans (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:
New tools are needed to simultaneously quantify and sequence ribonucleic acids (RNAs) to identify changes in expression levels and post-transcriptional modifications. Our lab previously developed a MALDI-based method to perform relative quantification on RNAs using an enzyme-mediated isotope labeling approach. Isotope labeling is made possible during the enzymatic digestion of intact RNAs in the presence of H₂¹⁸O. Control samples are similarly labeled during digestion in H₂¹⁶O. Decreasing sample size and increasing sample complexity both pose problems for the MALDI-based method that may be solved by switching to LC-MS. To implement this approach, digestions are combined prior to analysis. Standard oligonucleotide buffers are used for HPLC with triethylamine and 1,1,1,3,3,3-hexafluoroisopropanol as ion-pairing agents. Data-dependent MS/MS provides sequence information for RNA identification. We have determined the collision energies and mass ranges appropriate for the instrument; current investigations to improve sequencing includes building library databases for CID fragments and broadening the parent ion window to pass both the ¹⁶O and ¹⁸O labeled oligonucleotides. We have found that the presence of the isotopic label does not affect oligonucleotide retention time; thus mixtures of ¹⁶O and ¹⁸O labeled oligonucleotides of the same sequence will co-elute. The relative difference in the two samples can be accurately determined over one order of magnitude. The back-exchange of ¹⁸O labeled products to ¹⁶O labeled products was also investigated to increase the accuracy and precision of this tool for both MALDI and LC-MS. The effects of time, temperature, and pH on the digestion products were studied as a possible causes of back-exchange. We anticipate that this labeling approach, in combination with RNA sequencing, will provide a powerful technique for the simultaneous quantification and identification of RNAs at throughput levels significantly greater than presently available using standard biochemical methods.

Keywords: Mass Spectrometry, High Performance Liquid Chromatography, Quantitation