Development of Chemical Proteomic Methods to Quantify Cysteine Oxidation and Reactivity

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  • McConnell, Evan
    • College of Arts and Sciences, Department of Chemistry
Abstract
  • Protein oxidation is not a fully-understood concept in biology, where the identity of proteins containing oxidative post-translational modifications, their specific sites of modification, and the extent of changing oxidation under stress remain largely undefined. A major contributor to the cellular oxidative response is reversible oxidation of the sulfur atom on cysteine residues (i.e., protein thiols), which can modulate the structure and/or function of modified proteins and ultimately perturb metabolic and/or signaling pathways. Measuring the in vivo oxidation status of protein thiols has traditionally been challenging because these modifications are labile, prone to artifactual oxidation during sample preparation, and low in abundance. The fundamental goals of this dissertation are to develop chemical strategies to enrich for proteins thiols (Chapter 2) and determine their abundance using liquid chromatography-tandem mass spectrometry and label-free quantification (Chapter 3). Using the developed techniques, the proteome of the unicellular green alga Chlamydomonas reinhardtii was surveyed for proteins with reversibly oxidized cysteines and also phosphorylation sites to reveal a more complete understanding of PTM crosstalk, which highlights the reciprocity of signaling cascades across biological pathways (Chapter 4). The cysteine reactivity in the Chlamydomonas proteome was additionally measured to empirically determine sentinel proteins and define a characteristic fingerprint of biological response to oxidative stress (Chapter 5). As demonstrated, this dissertation provides novel tools to identify intracellular pathways targeted by protein thiol oxidation and are widely applicable in future proteomic studies using cells/tissues from disparate organisms. This work ultimately helps to measure cellular fitness by identifying novel markers of protein oxidation and provides targetable hypotheses to better understand tolerance mechanisms toward stress and disease.
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Advisor
  • Hicks, Leslie M.
  • Glish, Gary L.
  • Schoenfisch, Mark H.
  • Loeser, Richard F.
  • Ehrmann, Brandie M.
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2020
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