ANALYSIS OF RNA HYDROLYZATES BY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

This chapter discusses analysis of Ribo Nucleic Acid (RNA) hydrolyzates by liquid chromatography-mass spectrometry. Reversed-phase high-performance liquid chromatography (HPLC) is an experimentally effective technique for the separation of nucleosides, particularly for the analysis of enzymatic digests of RNA and Deoxyribo-nucleic acid (DNA). Depending on the problem at hand, identification of nucleoside constituents can often be made on the basis of retention times and Ultraviotet (UV) absorbance characteristics. As is common, in general, when chromatographic methods are used for purposes of identification, as opposed to simply for separation, the reliability of the method suffers as the complexity of the mixture increases, or when components of unknown or unexpected identity are encountered. The development of directly combined HPLC-mass spectrometry [liquid chromatography (LC)/MS] based on the thermospray interface provides a method which can be effectively applied to the analysis of nucleosides in nucleic acid digests, and is a powerful extension of the capabilities of either technique alone. Procedures for nuclease digestion of RNA to ribonucleotides, followed by dephosphorylation using alkaline phosphatase, are followed which, in general, permit direct injection of the crude digest directly into the HPLC. © 1990, Elsevier Inc. All rights reserved.