Characterization of O-Glycosylation and Phosphorylation on Nuclear Protein by Mass Spectrometry

Proteins are biomolecules that represent the ultimate expression of DNA, as they perform the majority of structural and functional roles in organisms. Proteins accomplish this through their interactions with other proteins and biomolecules, dictated by their molecular confirmation and location. A major regulator of protein activity is covalent post-translational modification (PTM). Characterizing protein PTMs is critical for developing a complete understanding of the proteome, the entire expressed protein complement of DNA. Over 400 PTMs have been identified, and liquid chromatography mass spectrometry (LCMS) has emerged as the most powerful technique to directly characterize protein PTMs. This dissertation reports the implementation of electron transfer dissociation (ETD) and collision-activated dissociation (CAD) in tandem with high-resolution MS to characterize O-linked glycosylation and phosphorylation PTMs on two nuclear proteins. Phosphorylation is a ubiquitous nucleocytoplasmic PTM that is found on Ser, Thr, and Tyr residues of thousands of metazoan proteins. It is involved in the regulation of a wide variety of cellular processes and is perhaps the mostly highly-studied PTM to date. Just 30 years ago, it was largely believed that nucleocytoplasmic protein glycosylation was non-existent. We now know that the nucleocytoplasmic monosaccharide, O-linked β–N-acetylglucosamine, or O-GlcNAc, modifies Ser and Thr residues of metazoan proteins from nearly every functional class, and it has a complex interplay with phosphorylation. O-GlcNAcylation has historically been challenging to study, but improvements in mass spectrometric techniques, such as the development of ETD that preserves labile O-linked PTMs like O-GlcNAc upon fragmentation, have significantly increased the ability to characterize this PTM. It is becoming increasingly evident that O-GlcNAcylation also regulates numerous cellular processes including transcription, translation, protein trafficking, and protein degradation.
Firstly, we report the comprehensive characterization of O-GlcNAcylation on Arabidopsis protein ‘repressor of ga1-3’, or RGA, a negative regulator of plant growth and development. This marks the first direct and comprehensive characterization of O-GlcNAcylation on a plant protein. In addition, numerous phosphorylation sites were discovered. Most notably and surprisingly, a previously unreported O-linked hexose monosaccharide PTM was discovered on RGA, representing only the second nucleocytoplasmic glyco-PTM reported to date.
Secondly, we report novel phosphorylation of S. cerevisiae DNA damage response mediator protein Rad9. Phosphorylation was detected on all 20 putative Cyclin-dependent kinase (CDK) motifs, including 7 previously unreported. All 9 CDK sites located in the Checkpoint kinase 1 (Chk1) activating domain (CAD) were phosphorylated and sites T125 and T143 were found to be especially important in facilitating Rad9-Chk1 interaction and Chk1 activation.