Quantification of the Proteins of the Bacterial Ribosome Using QconCAT Technology

Abstract

The bacterial ribosome is a complex of three strands of RNA and approximately 55 proteins. During protein synthesis, the ribosome interacts with other proteins, numbered in the hundreds, forming some stable and some transient complexes. The stoichiometries of these complexes and of partially assembled ribosomes are often unknown. We describe the development of a flexible standard for the determination of stoichiometries of ribosomal particles and complexes. A core QconCAT, an artificial protein consisting of concatenated signature peptides derived from the ribosomal proteins L2, L4, L13, S4, S7, and S8, was developed. The core QconCAT DNA construct incorporates restriction sites for the insertion of cassettes encoding signature peptides from additional proteins under study. Two cassettes encoding signature peptides from the remaining 30S and 50S ribosomal proteins were prepared, and the resulting QconCATs were expressed, digested, and analyzed by mass spectrometry. The majority of Escherichia coli ribosomal proteins are small and basic; therefore, tryptic digestion alone yields insufficient signature peptides for quantification of all of the proteins. The ribosomal QconCATs therefore rely on a dual-enzyme strategy: endoproteinase Lys-C digestion and analysis followed by trypsin digestion and further analysis. The utility of technology was demonstrated by a determination of the effect of gentamicin on the protein composition of the E. coli ribosome.