2D FTMS for Characterization of Modified RNA and Histones

In the nuclei of plant, animal, or human cells, DNA is wound around a complex system of special proteins called histones. When DNA is unwound for the cells to use it as a blueprint for protein synthesis, it is first transcribed into ribonucleic acid (RNA) that gets transported outside of the nucleus to code for proteins. Both histones and RNA are found to be chemically modified in vivo. These modifications have an influence on early development, inflammation, and neurodegeneration, but their precise role is yet to be understood. In order to understand the roles of these modifications in biological processes, it is important to be able to know the exact sequence of histones and RNA including their modifications, and to quantify how much each modification is represented in biological samples of RNA and histones. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is a powerful technique that measures the masses of intact ionized molecules. The electrically charged ions move through electric and magnetic fields, which bend their trajectories according to the ratio between their mass and their electric charge The trajectories are then measured by the FT-ICR MS and converted into the mass-to-charge ratios of the molecules. In addition, ions can be made to break apart into fragments by getting them to interact with neutral gas particles, photons, or electrons. FT-ICR MS can be used to determine the sequences of RNA and histones by measurement of fragment masses, to locate and identify any chemical modifications, and even to quantify their modifications. However, standard FT-ICR MS experiments can only sequence one molecule at a time. In biological samples, mixtures of RNA and histones can be difficult to separate, which necessitates new approaches for analysis of mixtures. Two-dimensional mass spectrometry (2D MS) is a highly promising technique that relies on periodic manipulation of the ions` position in the FT-ICR MS. With 2D MS, simultaneous sequencing of RNA or histones in mixtures and localizing and identifying their chemical modifications should become straightforward. In this project, we will develop 2D MS for the detailed analysis of modified RNA and histones, including the identification, localization, and relative quantitation of their modifications. The newly developed methodology will lay the foundation for future studies in which the biochemical function of RNA and histone modifications and their role in biological processes can be explored.

2D FT-ICR MS for the Structural Characterization of Modified RNA and Histones In the nuclei of plant and animal cells, DNA is wound around special proteins called histones. To serve as a blueprint for protein synthesis, DNA is first unwound and then transcribed into ribonucleic acid (RNA), which is transported out of the nucleus to code for proteins at the ribosomes. Both histones and RNA are chemically modified in vivo. These modifications have an influence on early development, inflammation, and neurodegeneration, but their precise role is yet to be understood. It is therefore important to determine the exact sequence of histones and RNA in biological samples including their modifications, and to determine how much and where exactly each modification is represented. Mass spectrometry is an analytical technique that is often used to sequence biomolecules. In biological samples, however, mixtures of RNA or histones can be difficult to separate, which necessitates new approaches for their analysis. Two-dimensional mass spectrometry (2D MS) can match fragments with precursors such that simultaneous sequencing of RNA or histone mixtures and the identification and localization of their chemical modifications becomes straightforward. A major challenge for 2D MS is the resolving power required for the characterization of larger biomolecules. After installing 2D MS at the University of Innsbruck, we thus focused on new approaches to overcome such limitations. Using mixtures of histone peptides with common modifications such as methylation and acetylation, we developed "narrowband" 2D MS to significantly increase the accuracy of 2D MS compared with regular "broadband" 2D MS, and demonstrated that this new technique is particularly suited for the analysis of large biomolecules such as histones and RNA. Further, we developed algorithms for "absorption mode" 2D MS, which improves data processing and doubles the spectral accuracy and thus the information obtained from 2D mass spectra. We demonstrated the performance of the combination of narrowband and absorption mode 2D MS for histone peptides with modifications that differ by as little as the mass of 10 electrons, and showed that fragments can be matched to the correct histone. By analysing different mixtures of histone peptides with different modifications, we showed for the first time that 2D MS can be used for the quantification of site-specific modifications of biomolecules. Moreover, we used the newly developed approaches for the characterization of modified RNA and ligand-bound RNA, and in collaboration with the Czech Academy of Sciences, applied 2D MS to the analysis of other modified proteins.