| Reversible protein phosphorylation on serine, threonine and tyrosine residues is one of the most common and important regulatory modifications of intracellular proteins, playing a role in many biological and biomedical phenomena such as cellular signal transduction, cell growth, cell differentiation, cell division, metabolism and cancer. Mass spectrometry has emerged as the method of choice for identifying phosphorylation sites in phosphopeptides because of its advantages over previous methods (high performance liquid chromatography separation of radiolabelled proteins with 32P or 33 P followed by Edman degradation) including its increased sensitivity and speed, and because it eliminates the need for protein radiolabelling.; This research focused on the application of mass spectrometry to phosphoproteomic analyses of Arabidopsis thaliana. We demonstrated the application of mass spectrometry to four phosphoproteomic projects. Before working on these projects, we examined the development of methods for phosphorylation enrichment and analysis. These methods were then applied to the various projects. Project 1, identifying autophosphorylation sites of a calcium-dependent protein kinase, demonstrated the use of several complementary methods for identifying numerous autophosphorylation sites of the protein. Project 2, identifying substrates of a calcium-dependent protein kinase from Arabidopsis thaliana, demonstrated the application of several newer technologies for identifying numerous substrates of the kinase. Project 3, identifying 14-3-3 interactors from Arabidopsis thaliana, examined the identification of numerous protein interactors, several of which were proven to be phosphorylated. These interactors were then shown to overlap with the substrates identified for the kinase, possibly indicating interaction between the two families of proteins. Finally, project 4, the application of robotic instrumentation was demonstrated as a means for high-throughput phosphoproteomic analysis, which resulted in identifying several phosphorylated proteins. |
