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Title: DOSCATs : double standards in quantitative proteomics
Author: Bennett, R. J. W.
ISNI:       0000 0004 7428 5950
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Since its inception, the field of proteomics has shifted from being a qualitative discipline, generating long lists of proteins within a sample, to a quantitative one, where how much of a protein is reported. With the advent of systems biology, the routine analysis of biomarker levels, and the requirement for robust, reliable data comparable between different laboratories, the importance of absolute quantification, where proteins are quantified in absolute titre, is becoming increasingly important. There are two commonly used techniques for absolute protein quantification, based on either mass spectrometry (MS) or immunochemical techniques such as western blotting (WB). MS is generally considered the gold standard technique for quantification, but WB can offer greater sensitivity and is much more accessible to researchers. Neither are intrinsically quantitative techniques and so rely on standards; either isotope labelled peptides or recombinant proteins bearing an epitope are used for MS or WB respectively. To improve the robustness and reproducibility of quantitative data it would be advantageous to apply both techniques for orthogonal quantification, but due to the very different calibration standards, workflows rarely overlap. DOSCATs (Double Standard conCATamers) are novel calibration standards that can unite MS and WB workflows, allowing for the quantification of direct comparison of quantitative data between the two platforms. DOSCATs, based on QconCAT technology, combine a series of epitope sequences concatenated with peptides in a single artificial protein. Stable isotope labelled peptide for MS analysis are released upon digestion with an enzyme such as trypsin, and intact DOSCATs act to bear multiple epitopes for WB. Also included were restricted proteolysis sites that allow for a mobility shift within WB, lending greater flexibility to the standard. The aim of this thesis was to develop and optimise the use of DOSCAT technology so that they could be used to quantify target proteins in both quantitative platforms. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. The DOSCAT was expressed and purified and the 9/13 peptides and 3/5 epitopes included in the sequence were observed by MS and WB respectively, demonstrating the proof of concept. However, restricted proteases performed poorly and three antibodies were discontinued by the manufacturer, so a second iteration of the NF-κB DOSCAT was designed. This was used to calibrate quantification by selected reaction monitoring MS (SRM-MS) and automated capillary WB. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. Building on this success, another DOSCAT was built for six proteins implicated to be indicative of paediatric Streptococcus pneumoniae meningitis infection. All six proteins were quantified by SRM-MS although QWB failed to quantify two targets as either DOSCAT or endogenous protein was not detected. SRM-MS data agreed very well with previous datasets generated for the same samples by label-free MS and QWB using full length standards, however, absolute values for DOSCAT calibrated QWB were inconsistent. This could be due to antibodies recognising DOSCAT and endogenous protein with different affinities. This work demonstrates that DOSCATs can be used as multiplexed, dual purpose standards to unite MS and WB workflows. The DOSCAT approach has the potential to generate reliable quantitative information particularly relevant for systems biology studies and contribute to the desired increase in reproducibility of biological research.
Supervisor: Beynon, Robert Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral