Phosphopeptide mapping of axon guidance molecules by Nano-ESI tandem mass spectrometry

Mass spectrometry has emerged as a powerful tool in the analysis of polypeptides. Improvements in ionization techniques have allowed tandem mass spectrometry experiments of proteins at biologically relevant levels. Tandem Mass spectrometry (MS/MS) allows for amino acid sequencing of peptides. This ability has expanded the use of MS as a bioanalytical tool beyond mass measurement into numerous areas including cataloguing of proteomes, identification of unknowns and more recently, the investigation of post-translational modifications including glycosylation and phosphorylation. In this thesis, I investigate the use of MS and MS/MS in the analysis of the tyrosine phosphorylation of selected axon guidance molecules. Phosphorylation of these molecules, including B-type Ephrins, Unc5/RCM and Robo, was confirmed and the site-specific locations of nine modified tyrosine residues were mapped by tandem mass spectrometry. These results formed the basis for targeted mutagenesis studies of the selected proteins to examine the physiological role of tyrosine phosphorylation in axon guidance.