Offline Two-Stage SEC and LC-MS/MS for Comprehensive Characterization of Yeast Ribosomal Populations

We report two-dimensional size exclusion chromatography tandem mass spectrometry (2D-SEC MS/MS), a workflow that couples two MS-compatible size exclusion chromatography steps to bottom-up proteomics for rapid, high-resolution characterization of Saccharomyces cerevisiae ribosomes. Building on the established Ribo Mega-SEC protocol, we show that a size exclusion chromatography (SEC) run on a 1000 Å column separates intact polysomes from mixed 80S/60S/40S particles, while a reinjection of the pooled 80S/60S/40S peak onto a second-dimension, 500 Å SEC column yields resolved monosome, 60S, and 40S subunit fractions. This method is thereby able to deliver four distinct ribosomal subpopulations in <90 min. Bottom-up LC-MS/MS of 2D-SEC fractions showed that our method can identify 78 of the 79-core yeast ribosomal proteins in the polysome fraction, with a 93% mean sequence coverage. It could also resolve and differentially quantify 36 of 38 paralogous protein pairs, revealing isoform-specific incorporation biases. Furthermore, our method could map all 12 known yeast ribosomal methylation sites and two canonical phosphorylation sites, enabling site-specific post-translational modification measurements. Applied to the study of ribosomes in nutrient deprivation and upon RPL7A knockout, we show that 2D-SEC MS/MS detects rapid polysome collapse and changes to the polysome profiling ratios, illustrating its capacity to link ribosome composition to functional perturbations.