Advertisement

2D-DIGE as a Tool in Neuroproteomics

  • Florian WeilandEmail author
Protocol
Part of the Neuromethods book series (NM, volume 146)

Abstract

Neuroproteomics encompasses the study of all protein-related dynamics of the nervous system, not only on a morphological level in development and disease but also functional aspects of the process of learning as well as changes in clinical conditions like depression and addiction. Detection of these changes in protein abundance under defined conditions is the field of differential proteome analysis, with two-dimensional difference gel electrophoresis (2D-DIGE) currently being the most comprehensive technique to quantify these changes while retaining information about isoforms and posttranslational modifications. This chapter focusses on technical aspects of 2D-DIGE as well as a brief overview of successful applications of this technique in neuroproteomics.

Keywords

2D-DIGE Proteomics 

Notes

Acknowledgments

The author wants to thank Maithili Shroff for proof-reading the manuscript.

References

  1. 1.
    Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18(11):2071–2077PubMedCrossRefGoogle Scholar
  2. 2.
    Alban A, David SO, Bjorkesten L, Andersson C, Sloge E, Lewis S, Currie I (2003) A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3(1):36–44PubMedCrossRefGoogle Scholar
  3. 3.
    Collier TS, Muddiman DC (2012) Analytical strategies for the global quantification of intact proteins. Amino Acids 43(3):1109–1117PubMedCrossRefGoogle Scholar
  4. 4.
    Kim MS, Pinto SM, Getnet D, Nirujogi RS, Manda SS, Chaerkady R, Madugundu AK, Kelkar DS, Isserlin R, Jain S, Thomas JK, Muthusamy B, Leal-Rojas P, Kumar P, Sahasrabuddhe NA, Balakrishnan L, Advani J, George B, Renuse S, Selvan LD, Patil AH, Nanjappa V, Radhakrishnan A, Prasad S, Subbannayya T, Raju R, Kumar M, Sreenivasamurthy SK, Marimuthu A, Sathe GJ, Chavan S, Datta KK, Subbannayya Y, Sahu A, Yelamanchi SD, Jayaram S, Rajagopalan P, Sharma J, Murthy KR, Syed N, Goel R, Khan AA, Ahmad S, Dey G, Mudgal K, Chatterjee A, Huang TC, Zhong J, Wu X, Shaw PG, Freed D, Zahari MS, Mukherjee KK, Shankar S, Mahadevan A, Lam H, Mitchell CJ, Shankar SK, Satishchandra P, Schroeder JT, Sirdeshmukh R, Maitra A, Leach SD, Drake CG, Halushka MK, Prasad TS, Hruban RH, Kerr CL, Bader GD, Iacobuzio-Donahue CA, Gowda H, Pandey A (2014) A draft map of the human proteome. Nature 509(7502):575–581PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Tazo Y, Hara A, Onda T, Saegusa M (2014) Bifunctional roles of survivin-DeltaEx3 and survivin-2B for susceptibility to apoptosis in endometrial carcinomas. J Cancer Res Clin Oncol 140:2027–2037CrossRefGoogle Scholar
  6. 6.
    Arentz G, Weiland F, Oehler MK, Hoffmann P (2015) State of the art of 2D DIGE. Proteomics Clin Appl 9(3–4):277–288PubMedCrossRefGoogle Scholar
  7. 7.
    Gorg A, Drews O, Luck C, Weiland F, Weiss W (2009) 2-DE with IPGs. Electrophoresis 30(Suppl 1):S122–S132PubMedCrossRefGoogle Scholar
  8. 8.
    Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F, Hawkins E, Currie I, Davison M (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1(3):377–396PubMedCrossRefGoogle Scholar
  9. 9.
    Shaw J, Rowlinson R, Nickson J, Stone T, Sweet A, Williams K, Tonge R (2003) Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3(7):1181–1195PubMedCrossRefGoogle Scholar
  10. 10.
    Sitek B, Potthoff S, Schulenborg T, Stegbauer J, Vinke T, Rump LC, Meyer HE, Vonend O, Stuhler K (2006) Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling. Proteomics 6(15):4337–4345PubMedCrossRefGoogle Scholar
  11. 11.
    Arnold GJ, Frohlich T (2012) 2D DIGE saturation labeling for minute sample amounts. Methods Mol Biol 854:89–112PubMedCrossRefGoogle Scholar
  12. 12.
    Weiland F, Zammit CM, Reith F, Hoffmann P (2014) High resolution two-dimensional electrophoresis of native proteins. Electrophoresis 35(12–13):1893–1902PubMedCrossRefGoogle Scholar
  13. 13.
    Schagger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199(2):223–231PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Altenhofer P, Schierhorn A, Fricke B (2006) Agarose isoelectric focusing can improve resolution of membrane proteins in the two-dimensional electrophoresis of bacterial proteins. Electrophoresis 27(20):4096–4111PubMedCrossRefGoogle Scholar
  15. 15.
    Zammit CM, Weiland F, Brugger J, Wade B, Winderbaum LJ, Nies DH, Southam G, Hoffmann P, Reith F (2016) Proteomic responses to gold(iii)-toxicity in the bacterium Cupriavidus metallidurans CH34. Metallomics 8(11):1204–1216PubMedCrossRefGoogle Scholar
  16. 16.
    Heinemeyer J, Scheibe B, Schmitz UK, Braun HP (2009) Blue native DIGE as a tool for comparative analyses of protein complexes. J Proteome 72(3):539–544CrossRefGoogle Scholar
  17. 17.
    Peters K, Braun HP (2012) Comparative analyses of protein complexes by blue native DIGE. Methods Mol Biol 854:145–154PubMedCrossRefGoogle Scholar
  18. 18.
    Reisinger V, Eichacker LA (2012) Native DIGE of fluorescent plant protein complexes. Methods Mol Biol 854:343–353PubMedCrossRefGoogle Scholar
  19. 19.
    Gillardon F, Rist W, Kussmaul L, Vogel J, Berg M, Danzer K, Kraut N, Hengerer B (2007) Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque deposition. Proteomics 7(4):605–616PubMedCrossRefGoogle Scholar
  20. 20.
    Mertins P, Yang F, Liu T, Mani DR, Petyuk VA, Gillette MA, Clauser KR, Qiao JW, Gritsenko MA, Moore RJ, Levine DA, Townsend R, Erdmann-Gilmore P, Snider JE, Davies SR, Ruggles KV, Fenyo D, Kitchens RT, Li S, Olvera N, Dao F, Rodriguez H, Chan DW, Liebler D, White F, Rodland KD, Mills GB, Smith RD, Paulovich AG, Ellis M, Carr SA (2014) Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels. Mol Cell Proteomics 13(7):1690–1704PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Espina V, Edmiston KH, Heiby M, Pierobon M, Sciro M, Merritt B, Banks S, Deng J, VanMeter AJ, Geho DH, Pastore L, Sennesh J, Petricoin EF 3rd, Liotta LA (2008) A portrait of tissue phosphoprotein stability in the clinical tissue procurement process. Mol Cell Proteomics 7(10):1998–2018PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Grassl J, Westbrook JA, Robinson A, Boren M, Dunn MJ, Clyne RK (2009) Preserving the yeast proteome from sample degradation. Proteomics 9(20):4616–4626PubMedCrossRefGoogle Scholar
  23. 23.
    Ahmed MM, Gardiner KJ (2011) Preserving protein profiles in tissue samples: differing outcomes with and without heat stabilization. J Neurosci Methods 196(1):99–106PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Kanshin E, Tyers M, Thibault P (2015) Sample collection method bias effects in quantitative Phosphoproteomics. J Proteome Res 14(7):2998–3004PubMedCrossRefGoogle Scholar
  25. 25.
    Smejkal GB, Rivas-Morello C, Chang JH, Freeman E, Trachtenberg AJ, Lazarev A, Ivanov AR, Kuo WP (2011) Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis. Electrophoresis 32(16):2206–2215PubMedCrossRefGoogle Scholar
  26. 26.
    Acosta-Martin AE, Chwastyniak M, Beseme O, Drobecq H, Amouyel P, Pinet F (2009) Impact of incomplete DNase I treatment on human macrophage proteome analysis. Proteomics Clin Appl 3(10):1236–1246PubMedCrossRefGoogle Scholar
  27. 27.
    Righetti PG, Chiari M, Gelfi C (1988) Immobilized pH gradients: effect of salts, added carrier ampholytes and voltage gradients on protein patterns. Electrophoresis 9(2):65–73PubMedCrossRefGoogle Scholar
  28. 28.
    Stark GR, Stein WH, Moore S (1960) Reactions of the Cyanate present in aqueous urea with amino acids and proteins. J Biol Chem 235(11):3177–3181Google Scholar
  29. 29.
    Rai AJ, Gelfand CA, Haywood BC, Warunek DJ, Yi J, Schuchard MD, Mehigh RJ, Cockrill SL, Scott GB, Tammen H, Schulz-Knappe P, Speicher DW, Vitzthum F, Haab BB, Siest G, Chan DW (2005) HUPO plasma proteome project specimen collection and handling: towards the standardization of parameters for plasma proteome samples. Proteomics 5(13):3262–3277PubMedCrossRefGoogle Scholar
  30. 30.
    Viswanathan S, Unlu M, Minden JS (2006) Two-dimensional difference gel electrophoresis. Nat Protoc 1(3):1351–1358PubMedCrossRefGoogle Scholar
  31. 31.
    Mujumdar RB, Ernst LA, Mujumdar SR, Waggoner AS (1989) Cyanine dye labeling reagents containing isothiocyanate groups. Cytometry 10(1):11–19PubMedCrossRefGoogle Scholar
  32. 32.
    Karp NA, Lilley KS (2005) Maximising sensitivity for detecting changes in protein expression: experimental design using minimal CyDyes. Proteomics 5(12):3105–3115PubMedCrossRefGoogle Scholar
  33. 33.
    Karp NA, Kreil DP, Lilley KS (2004) Determining a significant change in protein expression with DeCyder during a pair-wise comparison using two-dimensional difference gel electrophoresis. Proteomics 4(5):1421–1432PubMedCrossRefGoogle Scholar
  34. 34.
    Lilley KS, Friedman DB (2004) All about DIGE: quantification technology for differential-display 2D-gel proteomics. Expert Rev Proteomics 1(4):401–409PubMedCrossRefGoogle Scholar
  35. 35.
    Karp NA, McCormick PS, Russell MR, Lilley KS (2007) Experimental and statistical considerations to avoid false conclusions in proteomics studies using differential in-gel electrophoresis. Mol Cell Proteomics 6(8):1354–1364PubMedCrossRefGoogle Scholar
  36. 36.
    Buncel E, Symons EA (1970) The inherent instability of dimethylformamide-water systems containing hydroxide ion. J Chem Soc D 3:164–165CrossRefGoogle Scholar
  37. 37.
    Wang W, Ackermann D, Mehlich AM, Konig S (2011) Impact of quenching failure of cy dyes in differential gel electrophoresis. PLoS One 6(3):e18098PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Wang W, Ackermann D, Mehlich AM, Konig S (2010) False labelling due to quenching failure of N-hydroxy-succinimide-ester-coupled dyes. Proteomics 10(7):1525–1529PubMedCrossRefGoogle Scholar
  39. 39.
    Riederer IM, Herrero RM, Leuba G, Riederer BM (2008) Serial protein labeling with infrared maleimide dyes to identify cysteine modifications. J Proteome 71(2):222–230CrossRefGoogle Scholar
  40. 40.
    Qu Z, Meng F, Zhou H, Li J, Wang Q, Wei F, Cheng J, Greenlief CM, Lubahn DB, Sun GY, Liu S, Gu Z (2014) NitroDIGE analysis reveals inhibition of protein S-nitrosylation by epigallocatechin gallates in lipopolysaccharide-stimulated microglial cells. J Neuroinflammation 11:17PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Poschmann G, Grzendowski M, Stefanski A, Bruns E, Meyer HE, Stuhler K (2015) Redox proteomics reveal stress responsive proteins linking peroxiredoxin-1 status in glioma to chemosensitivity and oxidative stress. Biochim Biophys Acta 1854(6):624–631PubMedCrossRefGoogle Scholar
  42. 42.
    McNamara LE, Kantawong FA, Dalby MJ, Riehle MO, Burchmore R (2011) Preventing and troubleshooting artefacts in saturation labelled fluorescence 2-D difference gel electrophoresis (saturation DiGE). Proteomics 11(24):4610–4621PubMedCrossRefGoogle Scholar
  43. 43.
    Bjellqvist B, Ek K, Righetti PG, Gianazza E, Gorg A, Westermeier R, Postel W (1982) Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J Biochem Biophys Methods 6(4):317–339PubMedCrossRefGoogle Scholar
  44. 44.
    Righetti PG (1988) Isoelectric focusing as the crow flies. J Biochem Biophys Methods 16(2):99–110PubMedCrossRefGoogle Scholar
  45. 45.
    Olsson I, Larsson K, Palmgren R, Bjellqvist B (2002) Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension. Proteomics 2(11):1630–1632PubMedCrossRefGoogle Scholar
  46. 46.
    Altland K, Becher P, Rossmann U, Bjellqvist B (1988) Isoelectric focusing of basic proteins: the problem of oxidation of cysteines. Electrophoresis 9(9):474–485PubMedCrossRefGoogle Scholar
  47. 47.
    Görg A, Klaus A, Lück C, Weiland F, Weiss W (2007) Two-dimensional electrophoresis with immobilized pH gradients for proteome analysis: a laboratory manual. Technische Universität München, Freising-Weihenstephan, MünchenGoogle Scholar
  48. 48.
    Görg A, Postel W, Weser J, Günther S, Strahler JR, Hanash SM, Somerlot L (1987) Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer. Electrophoresis 8(2):122–124CrossRefGoogle Scholar
  49. 49.
    Westermeier R, Görg A (2011) Two-dimensional electrophoresis in proteomics. In: Protein purification. John Wiley & Sons, Inc., Hoboken, pp 411–439CrossRefGoogle Scholar
  50. 50.
    Morris JS, Clark BN, Wei W, Gutstein HB (2010) Evaluating the performance of new approaches to spot quantification and differential expression in 2-dimensional gel electrophoresis studies. J Proteome Res 9(1):595–604PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Morris JS, Clark BN, Gutstein HB (2008) Pinnacle: a fast, automatic and accurate method for detecting and quantifying protein spots in 2-dimensional gel electrophoresis data. Bioinformatics 24(4):529–536PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Sellers KF, Miecznikowski J, Viswanathan S, Minden JS, Eddy WF (2007) Lights, camera, action! Systematic variation in 2-D difference gel electrophoresis images. Electrophoresis 28(18):3324–3332PubMedCrossRefGoogle Scholar
  53. 53.
    Jung K, Gannoun A, Sitek B, Meyer HE, Stühler K, Urfer W (2005) Analysis of dynamic protein expression data. RevStat: Statist J 3(2):99–111Google Scholar
  54. 54.
    Dowsey AW, Dunn MJ, Yang GZ (2008) Automated image alignment for 2D gel electrophoresis in a high-throughput proteomics pipeline. Bioinformatics 24(7):950–957PubMedCrossRefGoogle Scholar
  55. 55.
    Dowsey AW, English J, Pennington K, Cotter D, Stuehler K, Marcus K, Meyer HE, Dunn MJ, Yang GZ (2006) Examination of 2-DE in the human proteome organisation brain proteome project pilot studies with the new RAIN gel matching technique. Proteomics 6(18):5030–5047PubMedCrossRefGoogle Scholar
  56. 56.
    Veeser S, Dunn MJ, Yang GZ (2001) Multiresolution image registration for two-dimensional gel electrophoresis. Proteomics 1(7):856–870PubMedCrossRefGoogle Scholar
  57. 57.
    Clark BN, Gutstein HB (2008) The myth of automated, high-throughput two-dimensional gel analysis. Proteomics 8(6):1197–1203PubMedCrossRefGoogle Scholar
  58. 58.
    Keeping AJ, Collins RA (2011) Data variance and statistical significance in 2D-gel electrophoresis and DIGE experiments: comparison of the effects of normalization methods. J Proteome Res 10(3):1353–1360PubMedCrossRefGoogle Scholar
  59. 59.
    Pursiheimo A, Vehmas AP, Afzal S, Suomi T, Chand T, Strauss L, Poutanen M, Rokka A, Corthals GL, Elo LL (2015) Optimization of statistical methods impact on quantitative proteomics data. J Proteome Res 14(10):4118–4126PubMedCrossRefGoogle Scholar
  60. 60.
    Storey JD, Tibshirani R (2003) Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 100(16):9440–9445PubMedPubMedCentralCrossRefGoogle Scholar
  61. 61.
    Storey JD (2002) A direct approach to false discovery rates. J R Statist SocB 64(3):479–498CrossRefGoogle Scholar
  62. 62.
    Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Statist SocB 57:289–300Google Scholar
  63. 63.
    Laeremans A, Van de Plas B, Clerens S, Van den Bergh G, Arckens L, Hu TT (2013) Protein expression dynamics during postnatal mouse brain development. J Exp Neurosci 7:61–74PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Pinaud R, Osorio C, Alzate O, Jarvis ED (2008) Profiling of experience-regulated proteins in the songbird auditory forebrain using quantitative proteomics. Eur J Neurosci 27(6):1409–1422PubMedPubMedCentralCrossRefGoogle Scholar
  65. 65.
    Simor A, Gyorffy BA, Gulyassy P, Volgyi K, Toth V, Todorov MI, Kis V, Borhegyi Z, Szabo Z, Janaky T, Drahos L, Juhasz G, Kekesi KA (2017) The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses. Mol Cell Neurosci 79:64–80PubMedCrossRefGoogle Scholar
  66. 66.
    Volgyi K, Udvari EB, Szabo ER, Gyorffy BA, Hunyadi-Gulyas E, Medzihradszky K, Juhasz G, Kekesi KA, Dobolyi A (2017) Maternal alterations in the proteome of the medial prefrontal cortex in rat. J Proteome 153:65–77CrossRefGoogle Scholar
  67. 67.
    Udvari EB, Volgyi K, Gulyassy P, Dimen D, Kis V, Barna J, Szabo ER, Lubec G, Juhasz G, Kekesi KA, Dobolyi A (2017) Synaptic proteome changes in the hypothalamus of mother rats. J Proteome 159:54–66CrossRefGoogle Scholar
  68. 68.
    Laskowska-Macios K, Nys J, Hu TT, Zapasnik M, Van der Perren A, Kossut M, Burnat K, Arckens L (2015) Binocular pattern deprivation interferes with the expression of proteins involved in primary visual cortex maturation in the cat. Mol Brain 8:48PubMedPubMedCentralCrossRefGoogle Scholar
  69. 69.
    Osorio C, Sullivan PM, He DN, Mace BE, Ervin JF, Strittmatter WJ, Alzate O (2007) Mortalin is regulated by APOE in hippocampus of AD patients and by human APOE in TR mice. Neurobiol Aging 28(12):1853–1862PubMedCrossRefGoogle Scholar
  70. 70.
    Voos W, Rottgers K (2002) Molecular chaperones as essential mediators of mitochondrial biogenesis. Biochim Biophys Acta 1592(1):51–62PubMedCrossRefGoogle Scholar
  71. 71.
    Volgyi K, Haden K, Kis V, Gulyassy P, Badics K, Gyorffy BA, Simor A, Szabo Z, Janaky T, Drahos L, Dobolyi A, Penke B, Juhasz G, Kekesi KA (2017) Mitochondrial proteome changes correlating with beta-amyloid accumulation. Mol Neurobiol 54(3):2060–2078PubMedCrossRefGoogle Scholar
  72. 72.
    Laramee ME, Smolders K, Hu TT, Bronchti G, Boire D, Arckens L (2016) Congenital Anophthalmia and binocular neonatal Enucleation differently affect the proteome of primary and secondary visual cortices in mice. PLoS One 11(7):e0159320PubMedPubMedCentralCrossRefGoogle Scholar
  73. 73.
    Gellen B, Volgyi K, Gyorffy BA, Darula Z, Hunyadi-Gulyas E, Baracskay P, Czurko A, Hernadi I, Juhasz G, Dobolyi A, Kekesi KA (2017) Proteomic investigation of the prefrontal cortex in the rat clomipramine model of depression. J Proteome 153:53–64CrossRefGoogle Scholar
  74. 74.
    Catherman AD, Skinner OS, Kelleher NL (2014) Top down proteomics: facts and perspectives. Biochem Biophys Res Commun 445(4):683–693PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life SciencesUniversity of DundeeDundeeUK

Personalised recommendations