Analysis of Biological and Synthetic Ribonucleic Acids by Liquid Chromatography−Mass Spectrometry Using Monolithic Capillary Columns

Abstract

Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) has been evaluated as a method for the fractionation and desalting of ribonucleic acids prior to their characterization by electrospray ionization mass spectrometry. Monolithic, poly(styrene-divinylbenzene)-based capillary columns allowed the rapid and highly efficient fractionation of both synthetic and biological ribonucleic acids. The common problem of gas-phase cation adduction that is particularly prevalent in the mass spectrometric analysis of ribonucleic acids was tackled through a combination of chromatographic purification and the addition of ethylenediaminetetraacetic acid to the sample at a concentration of 25 mmol/L shortly before on-line analysis. For RNA molecules ranging in size from 10 to 120 nucleotides, the mass accuracies were typically better than 0.02%, which allowed the characterization and identification of failure sequences and byproducts with high confidence. Following injection of a 500 nL sample onto a 60 x 0.2 mm column, the limit of detection for a 120-nucleotide ribosomal RNA transcript from Escherichia coli was in the 50-80 fmol range. The method was applied to the analysis of synthetic oligoribonucleotides, transfer RNAs, and ribosomal RNA. Finally, sequence information was derived for low picomole amounts of a 32-mer RNA upon chromatographic purification and tandem mass spectrometric investigation in an ion trap mass spectrometer. Complete series of fragment ions of the c- and y-types could be assigned in the tandem mass spectrum. In conclusion, IP-RP-HPLC using monolithic capillary columns represents a very useful tool for the structural investigation and quantitative determination of RNAs of synthetic and biological origin.