Fourier transform ion cyclotron resonance mass spectrometry as a high throughput affinity screen to identify RNA binding ligands

Abstract

The continuing evolution of analytical instrumentation based on high performance mass spectrometry has catalyzed the development of more sophisticated and broadly applicable schemes by which biological macromolecules can be interrogated. These schemes are increasingly employed to characterize not only the intrinsic properties of biomolecules such as molecular weight, amino acid or base sequence, and extent of post-translational modification, but also to study extrinsic properties of molecules such as interactions with other biological molecules including multimer formation, substrate–ligand binding, and molecular recognition. In this work we summarize the evolution of mass spectrometry-based analytical schemes employed to characterize non-covalent biological complexes and describe the development of a parallel high throughput screening (HTS) strategy to identify small molecules that bind RNA targets using ESI-FTICR. The MASS (multitarget affinity/specificity screening) assay takes advantage of the `intrinsic mass' label of each compound and target RNA by obtaining high resolution, high precision mass measurements of intact RNA–ligand complexes. The ability to analyze complex mixtures allows ligand mixtures to be screened in the presence of multiple RNA targets simultaneously. The identity of the small molecule(s) which bind, the RNA target to which it binds, and the compound-specific binding affinity can be determined in one set of rapid experiments.