PROJECT SUMMARY Transfer ribonucleic acids (tRNAs) are required for the synthesis of proteins within cells. tRNAs are found to contain the highest density and greatest diversity of post-transcriptionally modified nucleosides of any class of RNAs. Our scientific understanding of the functional role of these modified nucleosides in tRNAs is limited in large part by the lack of methods that can routinely identify and map modifications onto a primary tRNA sequence. The long-term goal of this research continues to be to develop appropriate mass spectrometric approaches that enable biological studies into the functional significance of modified tRNAs. This renewal is focused on the specific goal of identifying and understanding modified tRNAs from human samples. This research is timely and relevant as less than 15% of the nearly 270 predicted unique human tRNA sequences have been characterized at this level of detail. Moreover, a number of recent studies have implicated tRNAs and their modifications in a variety of different human diseases and pathologies. Our knowledge of the complete modification profiles of human tRNAs has been limited by the lack of bioanalytical methods capable of identifying specific modified ribonucleosides and particular sequence locations within individual tRNAs. Based on our advances in the last support period, the three specific aims in this renewal address the short-comings in the field by establishing a workflow that will define the population of tRNAs expressed in the sample, the census of modified nucleosides present, and the mapping of those modified nucleosides onto the specific expressed tRNAs. Our innovations will lead to the creation of tRNA region-specific modification profiling assays that can be used to track dynamic changes in these modifications across multiple sample types. We will demonstrate the utility and significance of our approach by examining changes in these profiles using melanocytes and various melanomas. This research plan will have significant impacts on multiple fields and disciplines. When complete, we will have an approach that can document the qualitative and quantitative differences of modified tRNA expressed in tissue, cell lines or disease condition. Our advances will provide the ability to develop insights into the role of tRNA modification status in protein synthesis regulation, proteostasis imbalances or disease state maintenance. Moreover, the bioanalytical developments to result from this research can be applied to scientific investigations that seek to understand how the cell regulates RNA modification patterns as well as how variable RNA modification patterns affect other cellular regulatory processes.