FT-ICR mass spectrometry of modified RNA and proteins

Mass spectrometry (MS) has become central to the identification of proteins by high- throughput peptide sequencing ("bottom-up" MS), whereas large-scale sequencing of ribonucleic acids (RNA) generally employs techniques other than MS. However, the recent realization that posttranscriptional modifications of non-coding RNA, and posttranslational modifications of proteins, play a key role in gene expression, demands even more sophisticated methodology with capabilities for the identification, localization, and relative quantitation of RNA and protein modifications. "Top-down" mass spectrometry holds great promise for this purpose as it can detect any alterations in RNA or protein mass, and potentially reveal any correlations that may exist between modifications. In this project, mechanistic studies on RNA and protein dissociation will be performed to lay the basis for the development and validation of new "top-down" and "middle-down" MS approaches for the comprehensive characterization of posttranscriptional and synthetic modifications of RNA, as well as posttranslational modifications of proteins. The new methodology using Fourier transform ion cyclotron resonance (FT-ICR) MS will be applied to select RNA and proteins of biological significance to reveal correlated editing in RNA, and combinatorial codes of proteins.

Ribonucleic acid (RNA) and protein modifications play an important role in the regulation of gene expression and the development of RNA-based therapeutics, but their identification, localization and relative quantitation by conventional biochemical methods can be quite challenging. A promising alternative are mass spectrometry (MS) based approaches that involve RNA or protein dissociation in the gas phase. For the development of such methods, it is essential to understand the dissociation mechanisms of unmodified, posttranscriptionally, posttranslationally, or synthetically modified RNA and proteins. In this project, we have performed mechanistic studies on RNA and protein dissociation and investigated the effect of a large variety of modifications on RNA and protein backbone bond cleavage. Our studies of peptides resolved a decades-old controversy on the mechanism of electron capture dissociation by showing that the positive charge responsible for peptide or protein backbone bond cleavage cannot generally be a sodium or other metal ion instead of a proton. Moreover, we demonstrated that MS can be used for the identification, localization and relative quantitation of biologically relevant nucleobase modifications, and our studies revealed an intrinsic preference for specific nucleobase-phosphate interactions in gaseous RNA ions that are highly similar to those found in self-cleaving RNA in solution (e.g., ribozymes).