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Quantification of the Proteins of the Bacterial Ribosome Using QconCAT Technology

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Manchester Institute for Biotechnology, 131 Princess Street, Manchester M1 7DS, United Kingdom
Manchester Pharmacy School, University of Manchester, Manchester M13 9PT, United Kingdom
§ Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom
*Phone: +44 (0) 161 275 2369. Fax: +44 (0) 161 275 2396. E-mail: [email protected]
Cite this: J. Proteome Res. 2014, 13, 3, 1211–1222
Publication Date (Web):February 4, 2014
https://doi.org/10.1021/pr400667h
Copyright © 2014 American Chemical Society

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    Abstract

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    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.

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    Numbers of peptides resulting from theoretical digests of ribosomal proteins using Protein Prospector and various proteolytic enzymes; DNA sequence of the synthetic gene (core QconCAT); core QconCAT sequence showing Lys-C peptides, tryptic peptides, and sacrificial peptides; SDS-PAGE showing expression in E. coli; MALDI mass spectrum of Lys-C digest of the core QconCAT before and after guanidination; MALDI mass spectrum of the sequential Lys-C/tryptic digest of the core QconCAT; core ribosomal QconCAT showing origins of the proteolytic peptides; expression of unlabeled 30S QconCAT in LB medium, unlabeled QconCAT in minimal medium, and labeled QconCAT in minimal medium; signature peptides in the 30S ribosomal subunit QconCAT; 50S ribosomal QconCAT sequence showing Lys-C peptides, tryptic peptides, and sacrificial peptides containing restriction enzymes; expression of labeled 50S QconCAT in minimal medium; expression of the 30S and 50S QconCAT in the total and soluble fractions of transformed E. coli cells; His-tag purification of the 50S-labeled QconCAT; LC–MS/MS Mascot results from a single LC–MS/MS analysis of a Lys-C digest and a tryptic digest of the 50S ribosomal QconCAT using a LTQ-Orbitrap mass spectrometer XL; MALDI-TOF mass spectra of 50S QconCAT labeled Lys-C digest; 50S ribosomal QconCAT peptides; and Q-TOF CID MS/MS spectra of the doubly protonated peptides ion, TYAAITGAEGK (S6), at m/z 544.24. This material is available free of charge via the Internet at http://pubs.acs.org.

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