Analysis of Post-translationally Modified Proteins by Mass Spectrometry

This dissertation describes three projects that were performed in collaboration with Tai-Ping Sun’s lab at Duke University. In each project, mass spectrometry was used to characterize the post-translational modification of proteins involved in the control of plant growth. The particular focus of these experiments was the identification of O-Fucosylated, O-GlcNAcylated, and phosphorylated proteins by mass spectrometry and the determination of the modification sites on those proteins by electron transfer dissociation. In the first chapter, samples of DELLA protein RGA purified from Arabidopsis plants with mutations to the O-GlcNAc transferase SEC and the protein O-Fucosyltransferase SPY were analyzed by mass spectrometry and electron transfer dissociation. RGA is a negative transcriptional regulator involved in the gibberellin signaling pathway. This work confirmed that RGA is oppositely regulated by SPY and SEC in its native Arabidopsis, with O-Fucosylation enhancing plant growth and O-GlcNAcylation repressing plant growth. Additionally, several modification sites were unambiguously determined.
In the second chapter of this dissertation, 22 proteins from Arabidopsis and 56 proteins from tobacco were identified with varying levels of O-Fucosylation using lectin affinity enrichment and mass spectrometry. Aleuria aurantia lectin was used to enrich O-Fucosylated peptides from tryptic digest mixtures produced from protein extracts of Arabidopsis and tobacco. An MS method employing a neutral loss trigger to preferentially select precursors bearing O-Fucosylation was used to unambiguously identify fucosylated proteins by direct detection of the modified peptide(s). The results identified proteins involved in multiple mechanisms of control of plant growth bearing O-Fucosylation, expanding the limited understanding of the global role of fucosylation in plants.
The final chapter describes the LC-MS/MS analysis of two auxin response factor proteins, ARF6 and ARF8, purified from transgenic tobacco plants with co-expression of SPY and SEC. These proteins are transcriptional regulators that act downstream of the plant hormone auxin. The results confirm that ARF6 and ARF8 are extensively modified by both enzymes, confirming an additional method of control of plant growth by nuclear O-Fucosylation and O-GlcNAcylation: modulation of the auxin signaling pathway.