Characterization of complex mixtures of isomeric oligonucleotides by electrospray ionization tendem mass spectrometry

The research presented here explores the boundaries to which electrospray ionization mass spectrometry can be extended in the analysis of isomeric nucleic acids. Methods of analysis and guidelines for interpretation of the complex collision-induced dissociation mass spectra derived from unfractionated mixtures of isobaric oligonucleotides are first presented as a foundation for the subsequent work. Methods and techniques were developed for the detection and sequence analysis of oligonucleotides containing pseudouridine, the most abundant of the modified nucleosides in nature, and the only one of more than 100 known modifications that does not present a mass shift from its underlying, unmodified precursor. These methods do not require any chemical derivatization, nor other specialized instrumentation. Finally, a novel assay was developed that utilizes liquid chromatography and tandem mass spectrometry with two stages of dissociation to determine the presence of pseudouridylated oligonucleotides in complex mixtures. Nanospray tandem mass spectrometry of isolated fractions, in concert with a gene sequence, enabled the primary structure determination of pseudouridine in the context of large RNAs (?1500 nt).