Search

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

Ribosome biogenesis involves numerous pre-rRNA processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Biedka et al. show that ribosomal proteins and assembly factors remodel several neighborhoods, including two 60S ribosomal subunit functional centers, during removal of the ITS2 spacer RNA., Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C2 site of 27SB pre-rRNA. C2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C2 cleavage and interpreted these results using cryo–electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C2 cleavage. Interestingly, when C2 cleavage is directly blocked by depleting or inactivating the C2 endonuclease, assembly progresses through all other subsequent steps.

A major gap in our understanding of ribosome assembly is knowing the precise function of each of the ∼200 assembly factors. The steps in subunit assembly in which these factors participate have been examined for the most part by depleting each protein from cells. Depletion of the assembly factor Erb1 prevents stable assembly of seven other interdependent assembly factors with pre-60S subunits, resulting in turnover of early preribosomes, before the ITS1 spacer can be removed from 27SA3 pre-rRNA. To investigate more specific functions of Erb1, we constructed eight internal deletions of 40–60 amino acid residues each, spanning the amino-terminal half of Erb1. The erb1Δ161–200 and erb1Δ201–245 deletion mutations block a later step than depletion of Erb1, namely cleavage of the C2 site that initiates removal of the ITS2 spacer. Two other remodeling events fail to occur in these erb1 mutants: association of twelve different assembly factors with domain V of 25S rRNA, including the neighborhood surrounding the peptidyl transferase center, and stable association of ribosomal proteins with rRNA surrounding the polypeptide exit tunnel. This suggests that successful initiation of construction of these functional centers is a checkpoint for committing to spacer removal.