title: Quantitative label-free redox proteomics of reversible cysteine oxidation in red blood cell membranes.
abstract: Reversible oxidation of cysteine residues is a relevant posttranslational modification of proteins. However, the low activation energy and transitory nature of the redox switch and the intrinsic complexity of the analysis render quite challenging the aim of a rigorous high-throughput screening of the redox status of redox-sensitive cysteine residues. We describe here a quantitative workflow for redox proteomics, where the ratio between the oxidized forms of proteins in the control vs treated samples is determined by a robust label-free approach. We critically present the convenience of the procedure by specifically addressing the following aspects: (i) the accurate ratio, calculated from the whole set of identified peptides rather than just isotope-tagged fragments; (ii) the application of a robust analytical pipeline to frame the most consistent data averaged over the biological variability; (iii) the relevance of using stringent criteria of analysis, even at the cost of losing potentially interesting but statistically uncertain data. The pipeline has been assessed on red blood cell membrane challenged with diamide as a model of a mild oxidative condition. The cluster of identified proteins encompassed components of the cytoskeleton more oxidized. Indirectly, our analysis confirmed the previous observation that oxidized hemoglobin binds to membranes while oxidized peroxiredoxin 2 loses affinity.
authors.affiliation: Department of Molecular Medicine, via A. Gabelli, 63, I-35121 Padova, Italy.
authors.forename: Mattia
authors.initials: M
authors.lastname: Zaccarin
authors.affiliation: Department of Molecular Medicine, via A. Gabelli, 63, I-35121 Padova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Falda
authors.affiliation: Department of Molecular Medicine, via A. Gabelli, 63, I-35121 Padova, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Roveri
authors.affiliation: Department of Molecular Medicine, via A. Gabelli, 63, I-35121 Padova, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Bosello-Travain
authors.affiliation: Department of Molecular Medicine, via A. Gabelli, 63, I-35121 Padova, Italy.
authors.forename: Luciana
authors.initials: L
authors.lastname: Bordin
authors.affiliation: Department of Molecular Medicine, via A. Gabelli, 63, I-35121 Padova, Italy.
authors.forename: Matilde
authors.initials: M
authors.lastname: Maiorino
authors.affiliation: Department of Molecular Medicine, via A. Gabelli, 63, I-35121 Padova, Italy.
authors.forename: Fulvio
authors.initials: F
authors.lastname: Ursini
authors.affiliation: Department of Molecular Medicine, via A. Gabelli, 63, I-35121 Padova, Italy. Electronic address: stefano.toppo@unipd.it.
authors.forename: Stefano
authors.initials: S
authors.lastname: Toppo
mesh_terms: D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D003599:Cytoskeleton / Q000737:chemistry; D003958:Diamide / Q000737:chemistry / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D054464:Peroxiredoxins / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*
chemical_list: D004220:Disulfides; D006454:Hemoglobins; D008565:Membrane Proteins; D003958:Diamide; C086065:PRDX2 protein, human; D054464:Peroxiredoxins; D003545:Cysteine
keywords: Cysteine residue oxidation; Disulfides; Red blood cell membrane; Redox proteomics