Top-down mass spectrometry of proteins and RNA

Today`s biomolecular research would be unthinkable without the contributions of modern mass spectrometry (MS). For protein and RNA identification and characterization by MS, two complementary strategies have evolved during the past decade: the "top-down" and "bottom-up" approaches. In the more commonly used bottom-up approach, the biomolecule of interest is first digested with enzymes in solution, and then the digestion products are analyzed by mass spectrometry. The top-down approach, on the other hand, analyzes intact biomolecular ions as well as their fragment ions from dissociation in the mass spectrometer. The mass spectrum of the intact ions gives immediate information on biomolecular heterogeneity and sequence errors, and characteristic differences between experimental and DNA-predicted mass values indicate specific post-translational or -transcriptional modifications (PTMs), e.g. 79.9663 Da for phosphorylation and 14.0156 Da for methylation. This biomolecular mass information is not available in the bottom-up MS approach. Moreover, a protein "fragment ladder" from top-down MS experiments can provide a complete description of a protein`s primary structure and reveal all of its modifications. Despite these significant advantages, top-down MS is only beginning to be more widely used. This is, in part, because the dissociation of gaseous biomolecular ions in the mass spectrometer turned out to be quite challenging, especially for larger ions: After transfer into the gas phase, the gaseous biomolecular ions can form compact structures that withstand the dissociation step. In this project, a new experimental strategy based on electron-ion interactions for biomolecular dissociation and post-charging for ion unfolding in top-down MS will be realized. Taking advantage of structural changes during the ion`s transition from solution to gas phase, this new approach will be applied to transiently unfolded biomolecular ions in the source region of the mass spectrometer. Because backbone cleavage and biomolecular conformation will be dealt with separately, thermal and collisional activation to prevent conformational collapse can be applied without exceeding the critical energy for loss of labile PTMs.