Characterization of Protein Sequences and Post-translational Modification by Mass Spectrometry

Kelley, Seamus, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Hunt, Donald, AS-Chemistry (CHEM), University of Virginia

This dissertation is comprised of three projects, in which mass spectrometry was used to characterize a unique collection of proteins. In the first project, multiple proteomic techniques were used to characterize the amino acid sequence of three monoclonal antibodies. Antibodies are large proteins that are produced by the immune system to recognize and bind a particular target with high specificity. The binding specific of each antibody is determined by the amino acid sequence in the variable domain. Specific and non-specific proteases were used to cleave each antibody into overlapping peptides, which were then de novo sequenced. The intact mass of each antibody subunit was determined through the digestion of the antibody hinge region. In-house modified instrumentation allowed for improvements in the intact MS/MS scans of intact subunits. Ultimately, each sequence was fully characterized and will be used to develop a bispecific immunoreagent to detect T cell secretion of cytokines in ex vivo tissue.

The second and third projects focused on characterizing post-translational modifications (PTMs) that may govern plant growth and cell signaling. Specifically, these projects describe new findings in the activity of two Arabidopsis thaliana glycosyltransferases, SECRET AGENT (SEC) and SPINDLY (SPY), which were previously known to oppositely regulate gibberellin signaling. We hypothesized that SEC and SPY target proteins that are involved in additional signaling pathways. Our work in transgenic tobacco showed that SEC and SPY modify two proteins involved in auxin signaling, Auxin Response Factors 6 and 8 (ARF6 and ARF8); and one protein involved in phosphate starvation, Phosphate Starvation Response 1 (PHR1). ARF6, ARF8, and PHR1 were purified from transgenic tobacco plants with co-expression of SPY and SEC, and samples with co-expression of SPY led to increased fucosylation, and samples with co-expression of SEC led to increased O-GlcNAcylation. Additionally, PTMs in purified ARF6 and ARF8 from wild type Arabidopsis were characterized. Several peptides in all samples were unambiguously site-localized using ETD.

PHD (Doctor of Philosophy)
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