Quantitative identification of non-coding RNAs by isotope labeling and LC-MS/MS

Non-protein coding ribonucleic acids (ncRNAs) are essential in biochemical processes in the cell including translation, protein regulation, and RNA modification. Of the various types of ncRNAs, transfer RNAs (tRNAs) have proven difficult to analyze and monitor individually in a complex mixture. Analysis of individual tRNAs provides insight into changes in ncRNA expression levels based on experimental and environmental changes. The goal of this research is to optimize both the isotopic labeling method and the liquid chromatography tandem mass spectrometry approaches to quantify changes in non-coding RNAs.;Quantification of RNA is achieved through endonuclease digestion and isotopic labeling of the 3'-terminus of the digestion products. The accuracy and precision of the relative quantification is dependent upon the efficiency of 18O incorporation and its integrity upon analysis. I have investigated the effect of pH, temperature, and enzyme presence on the removal of the 18O label as it is replaced with 16O over time using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS). Optimized conditions for sample handling and storage, which minimize back-exchange, should improve the accuracy and precision of results from this method.;Previous studies on the relative quantification of isotopically labeled RNAs have been performed utilizing MALDI-MS. I have employed the isotopic labeling method for use on a liquid chromatography tandem mass spectrometry (LC-MS/MS) platform; conditions that provide accurate and reproducible quantification of oligonucleotides have been investigated. The shift from a MALDI-MS to a LC-MS platform provides the ability for tandem mass spectral sequencing of RNAs and the analysis of more complex RNA mixtures.;The analysis of individual isoacceptors within the global tRNA pool is one specific biological system that these methods are developed to address. To monitor changes in expression levels on individual tRNA isoacceptors, the characterization of quantifiable signature digestion products on LC-MS/MS was investigated using total tRNA isolated from Escherichia coli. These verified quantifiable signature digestion products were then used to monitor changes in tRNA abundances from Escherichia coli cells grown under two different nutrient conditions. The methods described in this dissertation allow for the simultaneous identification and quantification of tRNAs under various environmental and experimental conditions.