Journal Article

Quantifying Reversible Oxidation of Protein Thiols in Photosynthetic Organisms

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

2015

Overview

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. Graphical Abstract ᅟ

• Publisher: Springer US,Springer Nature B.V,American Society for Mass Spectrometry
• Subject: Abundance / Analytical Chemistry / Arabidopsis - metabolism / Bioinformatics / Biotechnology / Chemistry / Chemistry and Materials Science / Chlamydomonas - metabolism / Chromatography, Liquid / Coefficient of variation / Fitness / Focus: Emerging Investigators: Research Article / Hydrogen peroxide / Mass spectrometry / Organic Chemistry / Oxidation / Oxidation-Reduction / Oxidative stress / Peptides / Photosynthesis / Plant Proteins - analysis / Plant Proteins - chemistry / Plant Proteins - metabolism / Proteins / Proteome - analysis / Proteome - chemistry / Proteome - metabolism / Proteomics / Proteomics - methods / Sulfhydryl Compounds - analysis / Sulfhydryl Compounds - chemistry / Sulfhydryl Compounds - metabolism / Tags / Tandem Mass Spectrometry / Thiols