Identification of modified nucleosides in intact transfer ribonucleic acid by pyrolysis-electron impact-collisional activation mass spectrometry

Abstract

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 comnnon 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 I -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.

Collision-induced dissociation of uracil and its derivatives

“…The conclusion [7,10] that the nucleoside-derived fragment ion (BHI) is structurally identical to the MH+ ion from the corresponding base was validated by showing that the CID mass spectra of several model pairs (e.g., uridine and uracil, 2-thiouridine and 2thiouracil) were virtually tndisttngutshable (data not shown). These results are in accord with earlier measurements carried out using odd-electron base ions formed by electron ionization, which showed analogous equivalence between base fragments and molecular ions of the free bases [22,23].…”