Quantifying reversible oxidation of protein thiols in photosynthetic organisms

William O. Slade, Emily G. Werth, Evan W. McConnell, Sophie Alvarez, Leslie M. Hicks

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Photosynthetic organisms use dynamic post-translational modifications to survive and adapt, which include reversible oxidative modifications of protein thiols that regulate protein structure, function, and activity. Efforts to quantify thiol modifications on a global scale have relied upon peptide derivatization, typically using isobaric tags such as TMT, ICAT, or iTRAQ that are more expensive, less accurate, and provide less proteome coverage than label-free approaches-suggesting the need for improved experimental designs for studies requiring maximal coverage and precision. Herein, we present the coverage and precision of resin-assisted thiol enrichment coupled to label-free quantitation for the characterization of reversible oxidative modifications on protein thiols. Using C. reinhardtii and Arabidopsis as model systems for algae and plants, we quantified 3662 and 1641 unique cysteinyl peptides, respectively, with median coefficient of variation (CV) of 13% and 16%. Further, our method is extendable for the detection of protein abundance changes and stoichiometries of cysteine oxidation. Finally, we demonstrate proof-of-principle for our method, and reveal that exogenous hydrogen peroxide treatment regulates the C. reinhardtii redox proteome by increasing or decreasing the level of oxidation of 501 or 67 peptides, respectively. As protein activity and function is controlled by oxidative modifications on protein thiols, resin-assisted thiol enrichment coupled to label-free quantitation can reveal how intracellular and environmental stimuli affect plant survival and fitness through oxidative stress.

Original languageEnglish (US)
Pages (from-to)631-640
Number of pages10
JournalJournal of the American Society for Mass Spectrometry
Volume26
Issue number4
DOIs
StatePublished - Apr 2015

Fingerprint

Sulfhydryl Compounds
Oxidation
Labels
Proteins
Proteome
Peptides
Resins
Oxidative stress
Post Translational Protein Processing
Algae
Arabidopsis
Stoichiometry
Design of experiments
Hydrogen Peroxide
Oxidation-Reduction
Cysteine
Oxidative Stress
Research Design

Keywords

  • Algae
  • Oxidative stress
  • Plants
  • Post-translational modifications
  • Quantitative proteomics
  • Reactive oxygen species
  • Redox proteomics

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

Cite this

Quantifying reversible oxidation of protein thiols in photosynthetic organisms. / Slade, William O.; Werth, Emily G.; McConnell, Evan W.; Alvarez, Sophie; Hicks, Leslie M.

In: Journal of the American Society for Mass Spectrometry, Vol. 26, No. 4, 04.2015, p. 631-640.

Research output: Contribution to journalArticle

Slade, William O. ; Werth, Emily G. ; McConnell, Evan W. ; Alvarez, Sophie ; Hicks, Leslie M. / Quantifying reversible oxidation of protein thiols in photosynthetic organisms. In: Journal of the American Society for Mass Spectrometry. 2015 ; Vol. 26, No. 4. pp. 631-640.
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