Protein Analysis by Mass Spectrometry

This dissertation describes two projects using mass spectrometry to analyze proteins. In project one we use LC-MS/MS and electron transfer dissociation to investigate the relationship between two Arabidopsis thaliana proteins, RGA and SPY, which are key plant growth regulators. SPY had previously been identified as an O-GlcNAc transferase that modified RGA, but our work led to the discovery that SPY is in fact a novel O-fucose transferase. Furthermore, we have established that O-GlcNAc and O-fucose modification have opposing effects on RGA. Modification with O-GlcNAc represses the growth inhibiting function of RGA, while O-fucose enhances it.
The second project describes the use of mass spectrometry to identify proteins that selectively bind to regions of DNA, called somatic hypermutation (SHM) enhancer regions, shown to be necessary for targeting SHM to the Immunoglobulin gene variable region in B cells. SHM is a key step in the immune system’s generation of high affinity antibodies, but how the mutational process is confined to the immunoglobulin locus is not yet understood. Using a label free quantitation strategy we identify a number of nuclear proteins from the DT40 and Ramos B cell lines, in particular the transcription factors Ikaros and Aiolos, that preferentially bind to enhancer DNA vs. control DNA.