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Title: Development and optimization of a workflow to enable mass spectrometry-based quantitative membrane proteomics of mature and tolerogenic dendritic cells
Author: Buck, Matthew Philip
ISNI:       0000 0004 6060 4930
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2014
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Tolerogenic dendritic cells are monocyte-derived dendritic cells (DC) cultured such that they adopt an immunoregulatory phenotype. In vitro, these cells are able to induce and maintain T cell tolerance through deviation of naive T cells to an anti-inflammatory phenotype and induction of anergy in memory T cells. Equivalent cells suppress established arthritis in murine models and tolerogenic DC are presently the subject of a phase I safety and efficacy trial at Newcastle University as part of the AutoDeCRA study. However, in spite of these promising data, we are yet to rigorously explore the basis of the phenotype of tolerogenic DC and lack markers to unequivocally distinguish them from other types of DC. This body of work is concerned with the development of a workflow to enable these questions to be addressed using mass spectrometry-based quantitative proteomics. Specifically, methods have been optimized and validated to enable a) enrichment and proteolytic digestion of membrane proteins, favouring their detection over more abundant cytoplasmic and nuclear proteins in LC/MS; b) differential stable isotope labelling of peptide N- and C-termini, enabling ‘isobaric peptide termini labelling’-based relative quantitation at the MS2 level; c) pipette-tip based anion exchange fractionation of IPTL-labelled peptides prior to LC/MS analysis, broadening depth of proteome coverage. Efforts to apply aspects of the workflow to perform quantitative comparisons of the whole cell proteomes and qualitative profiling of the membrane proteomes of mature and tolerogenic DC are also documented. It is envisaged that future application of this optimized workflow as a whole will enable the identification and relative quantitation of significant numbers of mature and tolerogenic DC plasma membrane proteins. Differentially expressed proteins of interest identified through this approach may then be further investigated for putative roles in tolerance induction.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available