Identification of Modified Nucleosides in Intact Transfer Ribonucleic Acid by Pyrolysis-electron Impact-collisional Activation Mass Spectrometry

A novel mass spectrometric method has been developed for the detection and identification of dihydrouridine, ribothymidine, 4-thiouridine, and 7-methylguanosine in Escherichia coli tRNAs. The method utilizes (a) Pyrolysis-Electron Impact-Mass Spectrometry (PYEIMS), a procedure which releases the purine and pyrimidine bases from the intact, underivatized tRNA molecule. The mass spectrum exhibits intense peaks for the bases deriving from the common nucleosides in tRNA as well as peaks of much lower intensity at mass values expected for the bases from modified components known to be present in the tRNA; and, (b) Collisional Activation Mass Spectrometry (CAMS), a technique which permits the isolation of a single ion species from a complex mass spectrum. Subsequent fragmentation of that species yields a characteristic collisional activation spectrum. Such analyses of the ion species that were presumed to originate from H2Urd, rThd, 4SUrd, and 7MeGuo in the tRNA were used to define the structure and, thus, the identity of each component. Attributes of the PYEICAMS technique are that (a) precise structural elucidation of minor nucleosides present in tRNAs at the 1 - 4% level is obtained; (b) the high order of sensitivity allows the analysis to be done on microgram amounts of tRNA; and (c) there is no requirement for enzymatic or chemical hydrolysis of the tRNA or for subsequent chromatographic separation methods.