HUPO, Sydney, Aus

Amy Claydon†, Ning Li*, Julian Selley°, Simon Hubbard°, John McCarthy*, Rob Beynon†
† = Proteomics and Functional Genomics group, Faculty of Health and Life Sciences, University of Liverpool
* = Structural and Functional Systems (MIB), Faculty of Life Sciences, University of Manchester

Associated with the 40S and 60S ribosomal subunit proteins are an unknown number of additional translational factors, responsible for regulating initiation and elongation during protein synthesis. Although the identity of many of these proteins is now known, it is timely to quantify these proteins, in cells and in association with functional ribosomes.
A targeted quantification strategy for ribosomal proteins was implemented using QconCAT technology. QconCATs are genes that encode concatenations of “quantotypic” peptides. In addition to the Q-peptides, each QconCAT includes a HisTag for purification, a short leader peptide that provides the initiator methionine, and common peptides at the N and C terminal (glufib and a minor variant thereof) for quantification of the standards. They are expressed heterologously in E. coli, in media containing stable isotope labelled amino acids and so can then be used as a labelled internal standard. Four QconCAT proteins were synthesised, mapping to 35 translational factors from Saccharomyces cerevisiae, including elongation factors, initiation factors and release factors.
A known amount of the expressed QconCAT protein added to a sample before proteolysis enables the absolute amount of the corresponding yeast proteins to be quantified. The QconCATs were added to different analytes: whole cell extracts, enriched fractions of 40S and 60S ribosomal subunits and salt washes obtained by treatment of purified ribosomes. For high sensitivity and selectively, proteins were quantified by selected reaction monitoring. Precursor/product transitions, obtained from MRM Atlas or by direct observation, were optimised for each Q-peptide. The QconCAT derived abundances were then compared to label-free and epitope-tagged data sets.