Multiplexed Mass Spectrometry of Modified RNAs

University Of Cincinnati Main Campus, Cincinnati OH

Investigators

Abstract

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Patrick Limbach and his group at University of Cincinnati are developing instrumental approaches based on mass spectrometry for the identification, sequencing and analysis of an important class of biomolecules, namely transfer ribonucleic acids (tRNAs). Because tRNAs are involved in the fundamental process of protein translation, it is important that new tools and approaches be developed that enable the sensitive and accurate determination of tRNA population and extent of modifications to the original tRNA sequence. This research will significantly advance the field of tRNA characterization by establishing the techniques required for multiplexed characterizations of tRNA populations. The advantage of multiplexed characterization is that significantly more information on tRNA identity and sequence modification can be obtained in a shorter time period and from less sample, as compared to current methods that analyze one tRNA at a time. This research project is multidisciplinary, involving the areas of chemistry, biochemistry and molecular biology, thus undergraduates and graduate students working on this project will gain key instruction and insights into the field of modern biological mass spectrometry. These activities continue a long-standing emphasis of Professor Limbach on expanding the number of engaged underrepresented minorities in science, in general, and mass spectrometry, in particular. During the award period, Professor Limbach and his group will develop two enzymatic approaches for tagging RNase digestion products with mass unique labels, which allow sample identity to be differentiated. These labeling strategies will overcome the limitations of existing 16O/18O-labeling approaches, are directly compatible with automated data analysis, and offer flexibility in sample purification or other sample manipulation steps. The labeling strategies include T4 RNA ligase, which will use nicotinamide adenine dinucleotide as the tag, and polyadenylate polymerase, which will use unmodified and modified dideoxynucleoside triphosphates as the tag. For each labeling approach, up to a four-plex comparative analysis assay is possible, which will enable information-rich investigations into a variety of biological processes. This new enzymatic labeling strategy for multiplexing our comparative analysis assay will be ideal for moving tRNA modification pattern analysis from bacteria into archaea and eukaryotes, including mammals. Because tRNA modification is known to be important in tRNA structure and function, these advances will provide researchers with a routine method for examining biological effects of hyper- and hypomodified tRNAs.