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Title: Analysis of the molecular components and phosphorylation of mouse brain proteomes
Author: Collins, M. O.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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We have developed upon existing immobilised metal-affinity chromatography (IMAC) techniques for capturing phosphopeptides, to selectively purify phosphoproteins from complex mixtures. Using this novel approach, combining both protein and peptide IMAC and MS data acquisition strategies, a comprehensive map of the mouse forebrain cytosolic phosphoproteome was achieved. This new approach was applied to purified mouse forebrain synaptosomes and resulted in the first large-scale map of the mouse synapse phosphoproteome. We have detected over 650 phosphorylation events, corresponding to 331 unique phosphorylation sites on synaptic proteins. 92% of these phosphorylation sites are novel, indicating a previously underestimated complexity in synaptic signalling. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest a small number of kinases phosphorylate many proteins and each substrate is phosphorylated by many kinases. In recent years, mass spectrometry (MS) based analysis of the postsynaptic density (PSD) and receptor complexes, have established for the first time a detailed list of its molecular components. In order to provide a coherent map of the molecular components of the synapse proteome, a bioinformatic approach was used to combine six PSD and three postsynaptic receptor complex datasets into a single database of synaptic proteins. This process of data integration allowed comparisons of analytical approaches used and revealed the most effective biochemical and MS-based methodologies. This data was used as a framework on which multiple data sources were integrated and allowed the derivation of proteome-wide molecular network maps at the level of gene regulation, protein interaction and protein phosphorylation. These maps were merged with functional or phenotype data from individual molecule studies to derive new models of synapse function.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available