Absolute quantitative proteomics typically relies on the use of stable isotope labelled internal standards introduced in a known amount. Comparative signal intensities of the labelled and unlabelled peptides allow the inference of protein concentration. However, the availability of standard labelled signature peptides in accurately known amounts is a limitation to the widespread of this approach. Here, new approach termed ''QconCAT'' has been developed for absolute quantification and for the determination of 30S and 50S ribosomal proteins stoichiometry. These QconCAT proteins are concatamers of Lys C/tryptic peptides for 54 ribosomal proteins. Trypsin, the most widely used enzyme in proteomics, has a few caveats especially with ribosomal proteins as it does not perform well due to the presence of high numbers of small and basic residue and creates relatively short peptides. There is, thus, room for improvement using alternative proteases. Here, we evaluate the performance of such a sequential strategy (Lys-C/Trypsin). We show this strategy, is capable of increasing number of suitable signature peptides for designing ribosomal proteins QconCATs. Secondly, the method for multiplexed absolute quantification, 30S QconCAT, has been extended to enable the determination of stoichiometry of ribosomal proteins in control and gentamicin-treated cultures in a single experiment using LTQ-Orbitrap. Ribosomal samples were isolated from the whole cell lysate by sucrose density centrifugation, and the relevant fractions were pooled and analyzed.Specifically we targeted ribosomes with sub-lethal concentrations of gentamicin and compared that with ribosome untreated samples. Our results showed that the stoichiometry of 30S ribosomal proteins and 3 others from large subunit was measured to be close to 1:1 copy number per ribosome along with some unique variations, such as ribosomal proteins S1 with low copy number and S2 with high copy number as established in the literature. Among these, ribosomal protein S4, S7 and S8 were selected for investigations as these proteins are early-assembly proteins and our result confirmed the stoichiometry of these proteins in untreated samples are very similar. We also found there is a variation in up-regulation of ribosomal proteins in treated cultures, result from the gentamicin cell injury. The last three chapters of this thesis dealing with the appearance of E.coli and S.epidermidis bacterial resistant to gentamicin and azithromycin, and this problem has inspired extensive searches towards the goal of obtaining novel drug targets that have a synergistic effect with these two anti-ribosomal drugs with improved antibacterial activity and reduced toxicity. We use label-free quantitative proteomic analysis coupled with mass spectrometry to investigate how E.coli and S.epidermidis adapts to the presence of sub-lethal concentrations of gentamicin and azithromycin in aerobic and oxygen-limited cultures as gentamicin effect on both conditions. We found that gentamicin caused the up-regulation of proteins involved in translation such as L1, L10, S2, S1, S8 , S10 and L9 in E.coli cultures. Three ribosomal proteins were up-regulated by gentamicin in both aerobic and oxygen-limited cultures: L1, L10 and S2. Similar expression for azithromycin on E.coli was observed, up-regulation of proteins involved in translation such as ribosomal proteins L6 and L11, whereas expression patterns of gentamicin-treated cells on S.epidermidis were un-expected as proteins necessary for pathogenesis were up-regulated by gentamicin. Our data provide several ribosomal proteins as drug targets that could produce a synergistic effect with gentamicin and azithromycin.
|Date of Award||1 Aug 2013|
- The University of Manchester
|Supervisor||Jill Barber (Supervisor) & Paul Sims (Supervisor)|
Quantification of ribosomal proteins by mass Spectrometry: an approach to drug target identification.
Al-Majdoub, Z. (Author). 1 Aug 2013
Student thesis: Phd