title: Quantifying reversible oxidation of protein thiols in photosynthetic organisms.
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.
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
authors.forename: William O
authors.initials: WO
authors.lastname: Slade
authors.forename: Emily G
authors.initials: EG
authors.lastname: Werth
authors.forename: Evan W
authors.initials: EW
authors.lastname: McConnell
authors.forename: Sophie
authors.initials: S
authors.lastname: Alvarez
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Hicks
mesh_terms: D017360:Arabidopsis / Q000378:metabolism; D002696:Chlamydomonas / Q000378:metabolism; D002853:Chromatography, Liquid; D010084:Oxidation-Reduction; D010788:Photosynthesis; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D010940:Plant Proteins; D020543:Proteome; D013438:Sulfhydryl Compounds