Quantification of the Proteins of the Bacterial Ribosome Using QconCAT Technology

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.