2D-DIGE as a proteomic biomarker discovery tool in environmental studies with Procambarus clarkii Ricardo Fernández-Cisnal a , Miguel A. García-Sevillano b , José L. Gómez-Ariza b , Carmen Pueyo a , Juan López-Barea a , Nieves Abril a, ⁎ a Department of Biochemistry and Molecular Biology and Agrifood Campus of International Excellence (ceiA3), University of Córdoba, Severo Ochoa Building, Rabanales Campus, 14071 Córdoba, Spain. b Department of Chemistry and Materials Science, Faculty of Experimental Science and Agrifood Campus of International Excellence (ceiA3), University of Huelva, El Carmen Campus, 21007 Huelva, Spain HIGHLIGHTS • Biological responses were analyzed in P. clarkii captured at sites of Doñana Na- tional Park and its surroundings (SW Spain). • Mn, Zn and Cu contents highly correlat- ed in P. clarkii tissues and sediments, while Cd and As were less correlated. • The higher metal levels promoted oxi- dative stress, increasing lipid peroxida- tion and reversibly oxidized protein thiols. • Protein expression profiles were traced by 2D-DIGE and 68/82 spots showed >± 2-fold changes. • MS/MS analysis identified 30 non-re- dundant proteins that were confirmed by Western blotting. GRAPHICAL ABSTRACT abstract article info Article history: Received 15 July 2016 Received in revised form 17 January 2017 Accepted 18 January 2017 Available online xxxx Editor: D. Barcelo A 2D-DIGE/MS approach was used to assess protein abundance differences in the red swamp crayfish Procambarus clarkii from polluted aquatic ecosystems of Doñana National Park and surrounding areas with differ- ent pollution loads. Procambarus clarkii accumulated metals in the digestive glands and gills reflecting sediment concentrations. We first stated that, probably related to elements accumulation, pollution increased oxidative damage in P. clarkii tissues, as shown by the thiol oxidation status of proteins and MDA levels. In these animals, the altered redox status might be responsible for the deregulated abundance of proteins involved in cellular re- sponses to oxidative stress including protein folding, mitochondrial imbalance and inflammatory processes. In- terestingly, polluted P. clarkii crayfish also displayed a metabolic shift to enhanced aerobic glycolysis, most Science of the Total Environment xxx (2017) xxx–xxx Abbreviations: ACN, acetonitrile; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate; 2-DE, two-dimensional gel electrophoresis; DBR, Doñana Biological Reserve; 2D-DIGE, two-dimensional differential in gel electrophoresis; DNP, Doñana National Park; DTT, dithiothreitol; EDTA, ethylene diamine tetraacetic acid; GSH, reduced glutathione; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; 5-IAF, 5-iodoacetamidofluorescein; IAM, iodoacetamide; ICP-MS, inductively-coupled plasma-mass spectrometry; IEF, isoelectro-focusing; IPG, immobilized pH gradient; LC-MS, liquid chromatography-mass spectrometry; LDP,Lucio del Palacio; LOD, limit of detection; LPO, lipid peroxidation; MAT, Matochal stream; MALDI, matrix-assisted laser desorption ionization; MDA, malondialdehyde; PAR, Partido stream; PCA, principal component analysis; PMSF, phenyl-methyl- sulphonyl fluoride; PVDF, Polyvinylidene fluoride; PTFE, Polytetrafluoroethylene; PTM, post-translational modifications; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; RNS, reactive nitrogen species; ROS, reactive oxygen species; TBA, thiobarbituric acid; TBP, tributylphosphine; TFA, trifluoroacetic acid; TOF, time-of-flight. ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology and Agrifood Campus of International Excellence (ceiA3-UCO), University of Córdoba, Severo Ochoa Building, Rabanales Campus, 14071 Córdoba, Spain. E-mail address: bb1abdim@uco.es (N. Abril). STOTEN-21838; No of Pages 15 http://dx.doi.org/10.1016/j.scitotenv.2017.01.125 0048-9697/© 2017 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Please cite this article as: Fernández-Cisnal, R., et al., 2D-DIGE as a proteomic biomarker discovery tool in environmental studies with Procambarus clarkii, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.125