DIGE Analysis of Fish Tissues

  • Joanna Nynca
  • Mariola A. Dietrich
  • Andrzej Ciereszko
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1664)

Abstract

Two-dimensional difference gel electrophoresis (2D-DIGE) appears to be especially useful in quantitative approaches, allowing the co-separation of proteins of control samples from proteins of treatment/disease samples on the same gel, eliminating gel-to-gel variability. The principle of 2D-DIGE is to label proteins prior to isoelectric focusing and use three spectrally resolvable fluorescent dyes, allowing the independent labeling of control and experimental samples. This procedure makes it possible to reduce the number of gels in an experiment, allowing the accurate and reproducible quantification of multiple samples. 2D-DIGE has been found to be an excellent methodical tool in several areas of fish research, including environmental pollution and toxicology, the mechanisms of development and disorders, reproduction, nutrition, evolution, and ecology.

Key words

2D-DIGE Minimal labeling CyDye Fish Tissue 

References

  1. 1.
    Martyniuk CJ, Alvarez S, Denslow ND (2012) DIGE and iTRAQ as biomarker discovery tools in aquatic toxicology. Ecotoxicol Environ Saf 76:3–10CrossRefPubMedGoogle Scholar
  2. 2.
    Beckett P (2012) The basis in 2D DIGE. In: Cramer A, Westermeier R (eds) Difference gel electrophoresis (DIGE); methods and protocols, Methods in molecular biology, vol 854. Humana, Clifton, NJ, pp 9–19CrossRefGoogle Scholar
  3. 3.
    Arentz G, Weiland F, Oehler MK, Hoffmann P (2015) State of the art of 2D DIGE. Proteomics Clin Appl 9:277–288CrossRefPubMedGoogle Scholar
  4. 4.
    Rees BB, Andacht T, Skripnikova E, Crawford DL (2011) Population proteomics: quantitative variation within and among populations in cardiac protein expression. Mol Biol Evol 28:1271–1279CrossRefPubMedGoogle Scholar
  5. 5.
    Gonzalez EG, Krey G, Espineira M, Diez A, Puyet A, Bautista JM (2010) Population proteomics of the European hake (Merluccius merluccius). J Proteome Res 9:6392–6404CrossRefPubMedGoogle Scholar
  6. 6.
    Roland K, Kestemont P, Dieu M, Raes M, Silvestre F (2016) Using a novel “integrated biomarker proteomic” index to assess the effects of freshwater pollutants in European eel peripheral blood mononuclear cells. J Proteomics 137:83–96CrossRefPubMedGoogle Scholar
  7. 7.
    Wang YY, Wang DZ, Lin L, Wang MH (2015) Quantitative proteomic analysis reveals proteins involved in the neurotoxicity of marine medaka Oryzias melastigma chronically exposed to inorganic mercury. Chemosphere 119:1126–1133CrossRefPubMedGoogle Scholar
  8. 8.
    Wang MH, Wang YY, Zhang L, Wang J, Hong HS, Wang DZ (2013) Quantitative proteomic analysis reveals the mode-of-action for chronic mercury hepatotoxicity to marine medaka (Oryzias melastigma). Aquat Toxicol 130:123–131CrossRefPubMedGoogle Scholar
  9. 9.
    Dorts J, Kestemont P, Thezenas ML, Raes M, Silvestre F (2014) Effects of cadmium exposure on the gill proteome of Cottus gobio: modulatory effects of prior thermal acclimation. Aquatic Toxicol 154:87–96CrossRefGoogle Scholar
  10. 10.
    Dorts J, Kestemont P, Dieu M, Raes M, Silvestre F (2011) Proteomic response to sublethal cadmium exposure in a sentinel fish species, Cottus gobio. J Proteome Res 10:470–478CrossRefPubMedGoogle Scholar
  11. 11.
    Eyckmans M, Benoot D, Van Raemdonck GAA, Zegels G, Van Ostade XWM, Witters E, Blust R, De Boeck G (2012) Comparative proteomics of copper exposure and toxicity in rainbow trout, common carp and gibel carp. Comp Biochem Physiol D Genomics Proteomics 7:220–232CrossRefPubMedGoogle Scholar
  12. 12.
    Jiang JL, Wang XR, Shan ZJ, Yang LY, Zhou JY, Bu YQ (2014) Proteomic analysis of hepatic tissue of Cyprinus carpio L. exposed to cyanobacterial blooms in Lake Taihu, China. PLoS One 9:e88211. doi:10.1371/journal.pone.0088211 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Roland K, Kestemont P, Loos R, Tavazzi S, Paracchini B, Belpaire C, Dieu M, Raes M, Silvestre F (2014) Looking for protein expression signatures in European eel peripheral blood mononuclear cells after in vivo exposure to perfluorooctane sulfonate and a real world field study. Sci Total Environ 468:958–967CrossRefPubMedGoogle Scholar
  14. 14.
    Roland K, Kestemont P, Henuset L, Pierrard MA, Raes M, Dieu M, Silvestre F (2013) Proteomic responses of peripheral blood mononuclear cells in the European eel (Anguilla anguilla) after perfluorooctane sulfonate exposure. Aquat Toxicol 128-129:43–52CrossRefPubMedGoogle Scholar
  15. 15.
    Dorts J, Kestemont P, Marchand PA, D'Hollander W, Thezenas ML, Raes M, Silvestre F (2011) Ecotoxicoproteomics in gills of the sentinel fish species, Cottus gobio, exposed to perfluorooctane sulfonate (PFOS). Aquat Toxicol 103:1–8CrossRefPubMedGoogle Scholar
  16. 16.
    Zhang W, Liu Y, Zhang HX, Dai JY (2012) Proteomic analysis of male zebrafish livers chronically exposed to perfluorononanoic acid. Environ Int 42:20–30CrossRefPubMedGoogle Scholar
  17. 17.
    Softeland L, Petersen K, Stavrum AK, Wu T, Olsvik PA (2011) Hepatic in vitro toxicity assessment of PBDE congeners BDE47, BDE153 and BDE154 in Atlantic salmon (Salmo salar L.) Aquat Toxicol 105:246–263CrossRefPubMedGoogle Scholar
  18. 18.
    De Wit M, Keil D, Remmerie N, van der Ven K, van den Brandhof EJ, Knapen D, Witters E, De Coen W (2008) Molecular targets of TBBPA in zebrafish analysed through integration of genomic and proteomic approaches. Chemosphere 74:96–105CrossRefPubMedGoogle Scholar
  19. 19.
    Varo I, Rigos G, Navarro JC, del Ramo J, Calduch-Giner J, Hernandez A, Pertusa J, Torreblanca A (2010) Effect of ivermectin on the liver of gilthead sea bream Sparus aurata: a proteomic approach. Chemosphere 80:570–577CrossRefPubMedGoogle Scholar
  20. 20.
    Pierrard MA, Kestemont P, Delaive E, Dieu M, Raes M, Silvestre F (2012) Malachite green toxicity assessed on Asian catfish primary cultures of peripheral blood mononuclear cells by a proteomic analysis. Aquat Toxicol 114:142–152CrossRefPubMedGoogle Scholar
  21. 21.
    Pierrard MA, Kestemont P, Phuong NT, Tran MP, Delaive E, Thezenas ML, Dieu M, Raes M, Silvestre F (2012) Proteomic analysis of blood cells in fish exposed to chemotherapeutics: evidence for long term effects. J Proteomics 75:2454–2467CrossRefPubMedGoogle Scholar
  22. 22.
    Ponnudurai RP, Basak T, Ahmad S, Bhardwaj G, Chauhan RK, Singh RA, Lalwani MK, Sivasubbu S, Sengupta S (2012) Proteomic analysis of zebrafish (Danio rerio) embryos exposed to cyclosporine A. J Proteomics 75:1004–1017CrossRefPubMedGoogle Scholar
  23. 23.
    De Wit M, Keil D, van der Ven K, Vandamme S, Witters E, De Coen W (2010) An integrated transcriptomic and proteomic approach characterizing estrogenic and metabolic effects of 17 alpha-ethinylestradiol in zebrafish (Danio rerio). Gen Comp Endocrinol 167:190–201CrossRefPubMedGoogle Scholar
  24. 24.
    Douxfils J, Lambert S, Mathieu C, Milla S, Mandiki SNM, Henrotte E, Wang N, Dieu M, Raes M, Rougeot C, Kestemont P (2014) Influence of domestication process on immune response to repeated emersion stressors in Eurasian perch (Perca fluviatilis, L.) Comp Biochem Physiol A Mol Integr Physiol 173:52–60CrossRefGoogle Scholar
  25. 25.
    Douxfils J, Deprez M, Mandiki SNM, Milla S, Henrotte E, Mathieu C, Silvestre F, Vandecan M, Rougeot C, Melard C, Dieu M, Raes M, Kestemont P (2012) Physiological and proteomic responses to single and repeated hypoxia in juvenile Eurasian perch under domestication–clues to physiological acclimation and humoral immune modulations. Fish Shellfish Immunol 33:1112–1122CrossRefPubMedGoogle Scholar
  26. 26.
    Varo I, Navarro JC, Rigos G, Del Ramo J, Calduch-Giner JA, Hernandez A, Pertusa J, Torreblanca A (2013) Proteomic evaluation of potentiated sulfa treatment on gilthead sea bream (Sparus aurata L.) liver. Aquaculture 376:36–44CrossRefGoogle Scholar
  27. 27.
    Gomes RSM, Skroblin P, Munster AB, Tomlins H, Langley SR, Zampetaki A, Yin XK, Wardle FC, Mayr M (2016) “Young at heart”: regenerative potential linked to immature cardiac phenotypes. J Mol Cell Cardiol 92:105–108CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Chen K, Cole RB, Rees BB (2013) Hypoxia-induced changes in the zebrafish (Danio rerio) skeletal muscle proteome. J Proteomics 78:477–485CrossRefPubMedGoogle Scholar
  29. 29.
    Purushothaman S, Saxena S, Meghah V, Lakshmi MGM, Singh SK, Swamy CVB, Idris MM (2015) Proteomic and gene expression analysis of zebrafish brain undergoing continuous light/dark stress. J Sleep Res 24:458–465CrossRefPubMedGoogle Scholar
  30. 30.
    Gundel U, Kalkhof S, Zitzkat D, von Bergen M, Altenburger R, Kuster E (2012) Concentration-response concept in ecotoxicoproteomics: effects of different phenanthrene concentrations to the zebrafish (Danio rerio) embryo proteome. Ecotoxicol Environ Saf 76:11–22CrossRefPubMedGoogle Scholar
  31. 31.
    Perez AN, Oehlers L, Heater SJ, Booth RE, Walter RB, David WM (2012) Proteomic analyses of the Xiphophorus Gordon-Kosswig melanoma model. Comp Biochem Physiol C Toxicol Pharmacol 155:81–88CrossRefPubMedGoogle Scholar
  32. 32.
    Lokaj K, Meierjohann S, Schuetz C, Teutschbein J, Schartl M, Sickmann A (2009) Quantitative differential proteome analysis in an animal model for human melanoma. J Proteome Res 8:1818–1827CrossRefPubMedGoogle Scholar
  33. 33.
    Biales AD, Bencic DC, Flick RL, Blocksom KA, Lazorchak JM, Lattier DL (2011) Proteomic analysis of a model fish species exposed to individual pesticides and a binary mixture. Aquat Toxicol 101:196–206CrossRefPubMedGoogle Scholar
  34. 34.
    Jostrup R, Shen W, Burrows JTA, Sivak JG, McConkey BJ, Singer TD (2009) Identification of myopia-related marker proteins in tilapia retinal, RPE, and choroidal tissue following induced form deprivation. Curr Eye Res 34:966–975CrossRefPubMedGoogle Scholar
  35. 35.
    Oehlers LP, Perez AN, Walter RB (2007) Detection of hypoxia-related proteins in medaka (Oryzias latipes) brain tissue by difference gel electrophoresis and de novo sequencing of 4-sulfophenyl isothiocyanate-derivatized peptides by matrix-assisted laser desorption/ionization time-of-flight mass, spectrometry. Comp Biochem Physiol C Toxicol Pharmacol 145:120–133CrossRefPubMedGoogle Scholar
  36. 36.
    Damodaran S, Dlugos CA, Wood TD, Rabin RA (2006) Effects of chronic ethanol administration on brain protein levels: a proteomic investigation using 2-D DIGE system. Eur J Pharmacol 547:75–82CrossRefPubMedGoogle Scholar
  37. 37.
    Richard N, Silva TS, Wulff T, Schrama D, Dias JP, Rodrigues PML, Conceicao LEC (2016) Nutritional mitigation of winter thermal stress in gilthead seabream: associated metabolic pathways and potential indicators of nutritional state. J Proteomics 142:1–14CrossRefPubMedGoogle Scholar
  38. 38.
    Ghisaura S, Anedda R, Pagnozzi D, Biosa G, Spada S, Bonaglini E, Cappuccinelli R, Roggio T, Uzzau S, Addis MF (2014) Impact of three commercial feed formulations on farmed gilthead sea bream (Sparus aurata, L.) metabolism as inferred from liver and blood serum proteomics. Proteome Sci 12:44. doi:10.1186/s12953-014-0044-3 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Addis MF, Pisanu S, Preziosa E, Bernardini G, Pagnozzi D, Roggio T, Uzzau S, Saroglia M, Terova G (2011) 2D DIGE/MS to investigate the impact of slaughtering techniques on postmortem integrity of fish filet proteins. J Proteomics 75:3654–3664CrossRefGoogle Scholar
  40. 40.
    Terova G, Addis MF, Preziosa E, Pisanu S, Pagnozzi D, Biosa G, Gornati R, Bernardini G, Roggio T, Saroglia M (2011) Effects of postmortem storage temperature on sea bass (Dicentrarchus labrax) muscle protein degradation: analysis by 2-D DIGE and MS. Proteomics 11:2901–2910CrossRefPubMedGoogle Scholar
  41. 41.
    Hamza N, Silvestre F, Mhetli M, Ben Khemis I, Dieu M, Raes M, Cahu C, Kestemont P (2010) Differential protein expression profile in the liver of pikeperch (Sander lucioperca) larvae fed with increasing levels of phospholipids. Comp Biochem Physiol D Genomics Proteomics 5:130–137CrossRefPubMedGoogle Scholar
  42. 42.
    Castets MD, Schaerlinger B, Silvestre F, Gardeur JN, Dieu M, Corbier C, Kestemont P, Fontaine P (2012) Combined analysis of Perca fluviatilis reproductive performance and oocyte proteomic profile. Theriogenology 78:432–442CrossRefPubMedGoogle Scholar
  43. 43.
    Forne I, Castellana B, Marin-Juez R, Cerda J, Abian J, Planas JV (2011) Transcriptional and proteomic profiling of flatfish (Solea senegalensis) spermatogenesis. Proteomics 11:2195–2211CrossRefPubMedGoogle Scholar
  44. 44.
    Forne I, Agulleiro MJ, Asensio E, Abian J, Cerda J (2009) 2-D DIGE analysis of Senegalese sole (Solea senegalensis) testis proteome in wild-caught and hormone-treated F1 fish. Proteomics 9:2171–2181CrossRefPubMedGoogle Scholar
  45. 45.
    Lucitt MB, Price TS, Pizarro A, Wu W, Yocum AK, Seiler C, Pack MA, Blair IA, FitzGerald GA, Grosser T (2008) Analysis of the zebrafish proteome during embryonic development. Mol Cell Proteomics 7:981–994CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Dietrich MA, Dietrich GJ, Mostek A, Ciereszko A (2016) Motility of carp spermatozoa is associated with profound changes in the sperm proteome. J Proteomics 138:124–135CrossRefPubMedGoogle Scholar
  47. 47.
    Dietrich MA, Arnold GJ, Fröhlich T, Otte KA, Dietrich GJ, Ciereszko A (2015) Proteomic analysis of extracellular medium of cryopreserved carp (Cyprinus carpio L.) semen. Comp Biochem Physiol D Genomics Proteomics 15:49–57CrossRefPubMedGoogle Scholar
  48. 48.
    Nynca J, Arnold GJ, Fröhlich T, Ciereszko A (2015) Cryopreservation-induced alterations in protein composition of rainbow trout semen. Proteomics 15:2643–2654CrossRefPubMedGoogle Scholar
  49. 49.
    Dietrich MA, Arnold GJ, Nynca J, Fröhlich T, Otte KA, Ciereszko A (2014) Characterization of carp seminal plasma proteome in relation to blood plasma. J Proteomics 98:218–232CrossRefPubMedGoogle Scholar
  50. 50.
    Ciereszko A, Dietrich MA, Nynca J (2017) Fish semen proteomics–new opportunities in fish reproductive research. Aquaculture 472:81–92CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Joanna Nynca
    • 1
  • Mariola A. Dietrich
    • 1
  • Andrzej Ciereszko
    • 1
  1. 1.Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food ResearchPolish Academy of SciencesOlsztynPoland

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