Efstathiou, Georgios (2012) A hypothesis-driven proteomics study for the investigation of interactions between macrophage neuroendocrine-immune pathways. PhD thesis, University of Warwick.

Abstract

Bioinformatics and mass spectrometry (MS)-based proteomics are specialities that became an integral part of biological research following the human genome sequencing project whereas the area of molecular systems biology (MSB) later emerged as a new approach to decoding life. The practice of MSB has made important contributions in our understanding of complex biological processes, primarily through modelling of high-throughput gene expression measurements at the level of transcription. Proteomics techniques have been successfully applied for systematic protein identification, characterization of protein-protein interactions and post-translational modifications. Practical large-scale quantitation, however, has traditionally been a considerable analytical challenge for MS-based proteomics technologies and this has hindered the construction of models based on protein expression data. In recent years this picture has begun to change with the rebirth of multiple reaction monitoring (MRM) MS and the emergence of targeted proteomics, where accurate quantitative expression measurements are obtained in a rapid and cost-effective manner for pre-specified sets of biologically interesting proteins. We employed triple quadrupole (TQ) instrumentation for measuring the expression of key proteins involved in the execution and regulation of the in ammatory response. We also perturbed the response through activation of neuroendocrine pathways downstream of the corticotropin releasing hormone (CRH) transmembrane receptor (CRH-R), in an effort to reveal novel dynamic changes in protein expression induced by CRH. The information required for the facilitation of the targeted experiment was previously obtained from: i) several large-scale protein identification experiments which involved the utilisation of a recent two-dimensional liquid chromatography fractionation technique and ii) de novo MRM assay development based on public datasets. The time-series expression data generated were processed and analysed by in-house developed software tools and finally modelled in an attempt to deduce relevant regulatory protein networks. Although the biological importance of CRH, a major component of the physiological stress system, in the context of local in ammation has been highlighted before, yet, as in cases of other neuropeptides, its immunoregulatory role remains unclear. The MSB approach employed here has enabled us to suggest potentially important components of the CRH effect. In the near future, we anticipate that the targeted proteomics approach will be an important ingredient of MSB research.

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