spectrometry platforms are utilized. Many of these workflows, however, have focused on individual synthetic RNAs or RNAs isolated and purified from biological sources with relatively little heterogeneity compared to RNAs in biological samples. In addition, the sample requirements for MS/MS, typically requiring concentrations in the range of hundreds of nanomolar, can be out-of-step with the amount of a single RNA that can be effectively isolated from a biological source. To begin addressing these challenges in mass spectrometry workflows, we have recently reported a nanoscale on-line desalting platform for nucleic acids, offering fast clean-up in a manner suitable for high-throughput analysis of RNAs, overcoming some of the obstacles to MS analysis. This foundational study focused on analyzing a single, isolated tRNA species with isoforms that could be readily resolved in the m/z domain. While tRNA can be readily extracted from cells, there are a significant number of possible species and isoforms. tRNAs are further diversified by an array of post-transcriptional modifications and non-canonical nitrogenous bases frequently incorporated into tRNAs, presenting a substantial degree of heterogeneity for analysis by MS. Furthermore, lacking an effective orthogonal chromatographic separation method prior to on-line desalting for tRNAs -owing to their high hydrophilicity and relative homogeneity in sequence length (70-80 nt)resolution of individual tRNA isoforms from a tRNA extract in the m/z domain is challenging. To extend the utility of MS/MS characterization of tRNA isoforms, we have developed an MS/MS strategy employing two fragmentation methodologies to provide information on potential precursor species present in an MS/MS scan and characterization of the fragments produced.