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An investigation of the use of MudPIT for the separation and quantification of proteins for GM croip safety assessment
Project Code: G03019
University of London - Royal Holloway College
Robertson, F ;
School of Biological Sciences, Royal Holloway, University of London
Fraser, P; Halket, J
Despite advances in gel-based approaches to proteomic analysis, the technology remains unsuitable for rigorous safety assessment of GM plants. Limiting factors include the type of proteins that can be analysed (i.e. hydrophobic proteins are poorly detected), inadequate dynamic range and inaccurate quantification. Multidimensional protein identification technology (MudPIT) is an attractive alternative. The procedure has been shown to detect and quantify low abundance peptides and peptides with hydrophobic properties.
The aim of the project was to assess the potential of MudPIT as a quantitative procedure to ascertain protein perturbations arising from genetic manipulation. The first stage of the project aimed to optimise front end procedures to maximise proteome coverage by MudPIT. The second stage of the project applied both so called‘non-chemical’ label-free methods, i.e., stable isotope labelled standards) and ‘chemical’ labelling methods (e.g. iTRAQ) to evaluate protein quantification of GM plant material. The unique collection of GM material and plants available at RHUL were used to fully evaluate and validate the procedure.
The MudPIT workflow was successfully developed for different mass spectrometry platforms: ESI Q-TOF, Orbitrap and Linear Ion traps and MALDI-TOF/TOF. A ‘non chemical’ label-free method was used to quantify iTRAQ labelled peptides using MS3. MudPIT was used with iTRAQ to gain quantitative information for 150 protein perturbations in non-GM and GM tomato cultivars developed and grown at RHUL using the Agilent 6520, QTOF. In the azygous cultivar, 7 proteins showed significant difference
from the wild type; these were stress response proteins. In the GM Psy1 sense cultivar, 60 proteins were found to be perturbed. 59 proteins were down-regulated and one protein was found to be significantly elevated in Psy1 sense: abscisic acid stress ripening inhibitor protein 1. The gene product phytoene synthase from the intended genetic alteration in Psy1 sense was notably absent from the iTRAQ quantitative protein profiles using the QTOF.
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