Phosphoprotein proteomics

The goal of this research was to investigate the use of gallium(III) immobilized metal affinity chromatography (Ga(III) IMAC) as a tool in the global internal standard technique (GIST). The work in this thesis began with the establishment of Ga(III) IMAC as a viable tool that was specific in its ability to select phosphorylated peptides from a complex mixture of peptides. This technique was perfected and automated in the analysis of phosphorylated peptides from casein proteins and milk samples. The second phase of this research was to establish a labeling technique that allowed the monitoring of up and down regulation of phosphorylated proteins. Light and heavy acetate and the quaternary amine coding agent were noted as candidates for this process. Once the ability to monitor up and down regulation of phosphorylated proteins was perfected, a more complex system was addressed. Whole milk from a healthy cow and whole milk from a cow stricken with mastitis were analyzed to determine if there was a difference in the regulation of phosphorylated proteins. Numerous phosphorylated proteins were monitored and confirmed through sequencing with mass spectrometry. Finally, one of the most complex protein systems available was analyzed. Liver hepatocytes from rats exposed to saline injections or microcystin-LR were monitored for their cellular regulation of phosphorylated proteins. The analysis resulted in numerous phosphorylated proteins identified and monitored for up and down regulation. Therefore, Ga(III) IMAC is a viable tool in the utilization of GIST for monitoring the cellular regulation of proteins within a complex protein mixture