Identification of Nucleosides in Hydrolysates of Transfer RNA by High-Resolution Mass Spectrometry

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Abstract
High-resolution mass spectrometry was investigated as a technique for identification of modified nucleosides in unfractionated hydrolysates of [Escherichia coli] tRNA. The method is based on recognition of predetermined sets of exact mass values which are characteristic of individual nucleoside components. Mass spectra are photographically recorded in a non-time-resolved fashion, so that differing rates of nucleoside vaporization are of no consequence. Experimental parameters of sensitivity, spectrometer resolution and rates of vaporization were studied. Under typical conditions, 1-4 .mu.g of tRNA are hydrolyzed, converted to volatile trimethylsilyl derivatives, and mass spectra of the resulting mixture recorded during 3 min at resolution 20,000; 75-100% of the minor nucleosides are usually identified in a single recording. The method is complementary to conventional methods of identification which rely on chromatographic mobilities, and can in principle be generally applied to recognition of biologically or chemically modified bases in nucleic acid.