Characterization of large molecular machines, such as the ribosome, under a breadth of biological states to elucidate regulatory details remains challenging. Herein, a workflow combining complex-up native mass spectrometry (nMS) with infrared multiphoton dissociation (IRMPD) and top-down proteomics (TDP) was developed to enable the rapid and direct characterization of ribosome heterogeneity. Preferential unfolding and fragmentation of rRNA by IRMPD enabled proteoform-resolved characterization of E. coli ribosome heterogeneity across growth states revealing ribosomal protein (RP) heterogeneity in unprecedented detail. TDP characterization of isolated RPs enabled confident proteoform characterization and facile interpretation of the IRMPD spectra. Additionally, differences in proteoform relative abundances determined by complex-up nMS and TDP reveal proteoform specific changes in relative interaction strengths. This experimental framework paves the way to a more rapid understanding of the diverse regulatory mechanisms conferred by ribonucleoprotein heterogeneity, including the role of RP composition and post-translational modification (PTM) heterogeneity in modulation of translation efficiency and specificity.
Tennakoon et al. (2026) studied this question.