Ribosome assembly, which comprises the processing and folding of the transcribed pre-rRNA and the assembly of the ribosomal proteins on the pre-rRNA, has been extensively characterized in budding yeast with the composition and structure of many assembly intermediates having been determined. In comparison, analysis of ribosome assembly in humans lags behind, in part due to the lack of effective method to isolate human early nucleolar pre-ribosomal particles.
We have developed an efficient approach for the systematic characterization of cellular protein complexes using uHPLC-based Size Exclusion Chromatography (SEC), and have shown that very large cellular complexes, including ribosomes and polysomes, can be separated within 15 minutes with high reproducibility by this SEC-uHPLC method, which we term, “Ribo Mega-SEC”.
We show here that we can apply Ribo Mega-SEC to effectively separate human pre-ribosomal particles from nucleolar extracts (“Pre-Ribo Mega-SEC”). We have used Pre-Ribo Mega-SEC in conjunction with mass spectrometry-based quantitative proteomics to characterize human nucleolar pre-ribosomal particles.