Characterization of non-protein coding ribonucleic acids by their signature digestion products and mass spectrometry
Transfer RNA (tRNA) and ribosomal RNA (rRNA) are two major non-protein coding ribonucleic acids (ncRNAs) in cell. In addition to their housekeeping roles during protein synthesis, they also participate in other cellular activities. The main objective of this dissertation is to characterize these two classic ncRNAs by their signature digestion products and MALDI mass spectrometry.
The separation of biologically active, pure, specific tRNAs is difficult due to the overall similarity in secondary and tertiary structures of different tRNAs. Because prior methods do not facilitate high-resolution separations of the extremely complex mixture represented by a cellular tRNA population, global studies of tRNA characterization are rare. I have found that the enzymatic digestion of an individual tRNA by a ribonuclease will generate digestion products yields a set of unique or signature digestion products that ultimately enable the detection of individual tRNA from a total tRNA pool. The detection is facilitated by MALDI-MS. This facile method enables the individual identification of tRNA isoacceptors without requiring any purification steps. I also developed a new approach including multiple ribonucleases to increase tRNA detection where an RNA mixture is digested separately with three ribonucleases, RNase T1, RNase A, and RNase TA, which generate their own sets of signature digestion products. The digestion conditions of these three ribonucleases with E. coli and B. subtilis were optimized.
This signature digestion product-based detection technique has been extended to ^16O- and ^18O-isotope labeling for RNA quantification. I introduced two equations to overcome the interfering peak-related difficulty and was able to calculate the ion abundance ratios of those 1 Da overlapping peaks. My studies on a mixture of standard tRNAs and total tRNAs of E. coli grown in MOPS minimal and EZ rich defined medium found that this approach provided quantitative results from those complex samples at light-to-heavy ratio between 2.5:1 and 1:2.5 with MALDI-MS. Selection of an appropriate product ion in the MALDI spectra is crucial for accurate results. Preliminary work has also been done related to the quantification of large rRNAs of E. coli and to the detection of cytoplasmic and mitochondrial tRNAs in yeast S. cerevisiae.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:signature digestion product transfer rna non protein coding maldi ms e coli s cerevisiae
Date of Publication:01/01/2008