Tandem mass spectrometry for structure assignments of wye nucleosides from transfer RNA

The tricyclic wye nucleoside family of eight known members constitutes one of the most complex and interesting series of posttranscriptionally modified nucleosides in transfer RNA. The principal reaction paths represented in collision-induced dissociation mass spectra of wye bases and their analogs have been studied in order to determine those structural features that can be readily established by mass spectrometry. The main routes of fragmentation are determined by the presence vs. absence of an amino acid side chain at C-7 (1H-imidazo[1,2-a]purine nomenclature). The common methionine-related side chain is cleaved at two points, providing a ready means of establishing the presence and net level of side chain modification. For those molecules without a side chain, the initial reaction steps are characteristically controlled by the presence vs. absence of methyl at N-4, allowing determination of the methylation status of that site. In the latter case initial opening of the central (pyrimidine) ring, in analogy to the dissociation behavior of guanine, causes loss of identity of C-6/C-7 so that placement of a single methyl at either site is not possible. Subsequent complex reaction paths follow, which include loss of CO and sequential loss of two molecules of HCN.