Ion laboratory for the elucidation of macromolecular assembly heterogeneity

PROJECT SUMMARY The view of the ribosome as a static automaton is undergoing a paradigm shift to a heterogeneous molecular machine with specialized forms yielding a ‘ribosome code’ for translation regulation. However, the full extent of ribosome heterogeneity and its biological role remains largely unknown. Despite decades of study, this knowledge gap persists due to the lack of technologies that bridge traditional biophysical methods and high- resolution structure determination approaches. Consequently, it is technologically very challenging to decipher which specific ribosomal components are (1) present, (2) their modifications, and (3) their stoichiometry as a function of cell type, localization, and state. There is a critical need for novel top-down and native mass spectrometry approaches for direct, rapid, and extensive characterization of large, heterogeneous molecular machines, such as the ribosome, to fundamentally understand the mechanisms of biological function. The goal of the proposed work is to directly characterize and quantify intact ribosomal proteins using top- down proteomics and determine the exact set of components that assemble to form specialized ribosomes using native mass spectrometry. To achieve this goal, the foundational technologies must first be developed for (1) accurate mass determination, (2) efficient and informative dissociation, and (3) spectral decongestion techniques which will enable more complete characterization of the intact ribosome and its individual components. This research program will develop and integrate novel (1) gas-phase charge reduction reactions, (2) hybrid tandem mass spectrometry methods, and (3) ion mobility separations that will enable production and interpretation of information rich top-down proteomics and native mass spectrometry data. These advancements will enable rapid measurements of the intact ribosome, gas phase dissection of the intact ribosome, and characterization of the sequence and modification of the individual ribosomal components. These broadly applicable technologies will immediately enable previously intractable studies in a wide range of biological systems. Long term, development and dissemination of these foundational technologies will enable more complete characterization of ribosome heterogeneity, advances in basic understanding of biology, and discovery of novel therapeutic targets for human health issues ranging from antibiotic resistance to cancer.