Quantifying Reversible Oxidation of Protein Thiols in Photosynthetic Organisms

Abstract

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.

Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets

“…Following ZipTip clean‐up, peptides were dried down and resuspended in 20 μl of 0.1% TFA, 5% MeCN before separation via a 90‐min linear gradient from 95% H 2 O/5% MeCN/0.1% formic acid (FA) to 65% H 2 O/35% MeCN/0.1% FA via a NanoAcquity UPLC (Waters) using a C18 column (NanoAcquity UPLC 1.8 μm HSS T3, 75 ×250 mm). A TripleTOF 5600 (AB Sciex, Framingham, MA, USA) Q‐TOF was operated in positive‐ionization nanoelectrospray and high‐sensitivity mode for data acquisition as described previously (Slade et al ., ). In addition to the Supporting Information tables for MS datasets, the mass spectrometry proteomics data have been deposited to the ProteomeXChange Consortium via PRIDE partner repository(Vizcaíno et al ., ) identifier PXD007221.…”

Proteome-Wide Analysis of Cysteine Reactivity during Effector-Triggered Immunity

“…Col-0 and top1top2 mutants). Proteins were extracted using a Tris-buffered phenol method described in Slade et al (2015). Briefly, leaf tissue (0.2 g) was ground under liquid N to a fine powder before homogenization in 2 mL of buffer containing 50 mM Tris (pH 8), 1 mM EDTA, 0.9 M sucrose, protease inhibitors (EDTA-free; Roche), and 100 mM IAM before adding SDS to a 1% (v/v) final concentration.…”

“…Mass spectrometry has a great potential to allow the systematic exploration of the plant redoxome. Several recent studies report proteome-wide mining of hydrogen peroxide (H 2 O 2 )-sensitive cysteines in several plant species (Alvarez et al, 2011;Wang et al, 2012b;Muthuramalingam et al, 2013;Liu et al, 2014;Slade et al, 2015), and collectively, highlight the broad impacts of ROS on plant proteomes. However, the composition of the plant redoxome in response to plantproduced ROS, the identity of redox sensors, and their contribution to plant defense and adaptive pathways remain largely unknown.…”

The Plant PTM Viewer, a central resource exploring plant protein modifications. From site-seeing to protein function

“…Other plant protein modifications that have been identified by MS but rarely catalogued include cysteine oxidation and proteolytic processing. Oxidized cysteine sites have been detected in Arabidopsis and Chlamydomonas reinhardtii , following differential labeling of oxidized and reduced thiols (Liu et al, 2014, Slade et al, 2015). Proteolytically processed sites have been detected in Arabidopsis by enrichment of newly exposed protein N-termini via positional proteomics (Kleifeld et al, 2010, Venne et al, 2015), identifying substrates of the protease METACASPASE9 (Tsiatsiani et al, 2013), processing associated with chloroplast protein import (Köhler et al, 2015, Rowland et al, 2015) and N-end rule substrates (Zhang et al, 2015, 2018).…”