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Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

Nature Protocols volume 1, pages 1732–1742 (2006)Cite this article

Abstract

Quantitative proteomics is the workhorse of the modern proteomics initiative. The gel-based and MuDPIT approaches have facilitated vital advances in the measurement of protein expression alterations in normal and disease phenotypic states. The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). 2D-DIGE is based on direct labeling of lysine groups on proteins with cyanine CyDye DIGE Fluor minimal dyes before isoelectric focusing, enabling the labeling of 2–3 samples with different dyes and electrophoresis of all the samples on the same 2D gel. This capability minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching. This protocol can be completed in 3–5 weeks depending on the sample size of the experiment and the level of expertise of the investigator.

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Figure 1: Schematic of 2D-DIGE experiment.
Figure 2: Schematic of DeCyder DIA.
Figure 3: Schematic of DeCyder BVA.

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References

  1. Tannu, N. & Hemby, S.E. in Progress in Brain Research: Functional Genomics and Proteomics in the Clinical Neurosciences Vol. 158 (eds Hemby, S.E. & Bahn, S.) (Elsevier, Amsterdam, 2006).

    Google Scholar 

  2. Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).

    CAS  PubMed  Google Scholar 

  3. Tannu, N., Mash, D.C. & Hemby, S.E. Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims. Mol. Psychiatry, doi:10.1038/sj.mp.4001914 (2006).

  4. O'Farrell, P.H. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021 (1975).

    Article  CAS  PubMed  Google Scholar 

  5. Bjellqvist, B. et al. Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J. Biochem. Biophys. Methods 6, 317–339 (1982).

    Article  CAS  PubMed  Google Scholar 

  6. Shomo, R.E., II, Chandrasekaran, A., Marshall, A.G., Reuning, R.H. & Robertson, L.W. Laser desorption/fourier transform ion cyclotron resonance mass spectrometry: digoxin, digitoxin, and their reduced and sugar-hydrolyzed metabolites. Biomed. Environ. Mass Spectrom. 15, 295–302 (1988).

    Article  CAS  PubMed  Google Scholar 

  7. Wildenauer, D.B. et al. Analysis of cerebrospinal fluid from patients with psychiatric and neurological disorders by two-dimensional electrophoresis: identification of disease-associated polypeptides as fibrin fragments. Electrophoresis 12, 487–492 (1991).

    Article  CAS  PubMed  Google Scholar 

  8. Henzel, W.J. et al. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. USA 90, 5011–5015 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pappin, D.J., Hojrup, P. & Bleasby, A.J. Rapid identification of proteins by peptide-mass fingerprinting. Curr. Biol. 3, 327–332 (1993).

    Article  CAS  PubMed  Google Scholar 

  10. Jonscher, K.R. & Yates, J.R., III The quadrupole ion trap mass spectrometer —a small solution to a big challenge. Anal. Biochem. 244, 1–15 (1997).

    Article  CAS  PubMed  Google Scholar 

  11. Unlu, M., Morgan, M.E. & Minden, J.S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077 (1997).

    Article  CAS  PubMed  Google Scholar 

  12. Yates, J.R., III Database searching using mass spectrometry data. Electrophoresis 19, 893–900 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Tannu, N.S. et al. Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting. Anal. Biochem. 327, 222–232 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Anderson, L. & Seilhamer, J. A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18, 533–537 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Gygi, S.P., Rochon, Y., Franza, B.R. & Aebersold, R. Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 19, 1720–1730 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Roberts, G.C. & Smith, C.W. Alternative splicing: combinatorial output from the genome. Curr. Opin. Chem. Biol. 6, 375–383 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. Identification of novel genes coding for small expressed RNAs. Science 294, 853–858 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Lee, Y., Jeon, K., Lee, J.T., Kim, S. & Kim, V.N. MicroRNA maturation: stepwise processing and subcellular localization. EMBO J. 21, 4663–4670 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sontheimer, E.J. & Carthew, R.W. Silence from within: endogenous siRNAs and miRNAs. Cell 122, 9–12 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Freeman, W.M. & Hemby, S.E. Proteomics for protein expression profiling in neuroscience. Neurochem. Res. 29, 1065–1081 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Schrader, W. & Klein, H.W. Liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS): an early overview. Anal. Bioanal. Chem. 379, 1013–1024 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Douglas, D.J., Frank, A.J. & Mao, D. Linear ion traps in mass spectrometry. Mass Spectrom. Rev. 24, 1–29 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Nilesh, S. & Tannu, S.E.H. in Progress in Brain Research (ed. Bahn, S.E.H.a.S.) 45 (Elsevier, 2006).

    Google Scholar 

  26. Yates, J.R., III, Gilchrist, A., Howell, K.E. & Bergeron, J.J. Proteomics of organelles and large cellular structures. Nat. Rev. Mol. Cell Biol. 6, 702–714 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Van den Bergh, G., Clerens, S., Vandesande, F. & Arckens, L. Reversed-phase high-performance liquid chromatography prefractionation prior to two-dimensional difference gel electrophoresis and mass spectrometry identifies new differentially expressed proteins between striate cortex of kitten and adult cat. Electrophoresis 24, 1471–1481 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Molloy, M.P., Brzezinski, E.E., Hang, J., McDowell, M.T. & VanBogelen, R.A. Overcoming technical variation and biological variation in quantitative proteomics. Proteomics 3, 1912–1919 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Tannu, N. & Hemby, S.E. Quantitation in two-dimensional fluorescence difference gel electrophoresis: effect of protein fixation. Electrophoresis 27, 2011–2015 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tonge, R. et al. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1, 377–396 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Alban, A. et al. A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3, 36–44 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Knowles, M.R. et al. Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis. Proteomics 3, 1162–1171 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Hu, Y., Malone, J.P., Fagan, A.M., Townsend, R.R. & Holtzman, D.M. Comparative proteomic analysis of intra- and interindividual variation in human cerebrospinal fluid. Mol. Cell Proteomics 4, 2000–2009 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Karp, N.A., Griffin, J.L. & Lilley, K.S. Application of partial least squares discriminant analysis to two-dimensional difference gel studies in expression proteomics. Proteomics 5, 81–90 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Swatton, J.E., Prabakaran, S., Karp, N.A., Lilley, K.S. & Bahn, S. Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Mol. Psychiatry 9, 128–143 (2004).

    Article  CAS  PubMed  Google Scholar 

  36. Lee, I.N. et al. Identification of human hepatocellular carcinoma-related biomarkers by two-dimensional difference gel electrophoresis and mass spectrometry. J. Proteome Res. 4, 2062–2069 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Zhou, G. et al. 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Mol. Cell Proteomics 1, 117–124 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Brown, L.M. et al. Quantitative and qualitative differences in protein expression between papillary thyroid carcinoma and normal thyroid tissue. Mol. Carcinog. 45, 613–626 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Nakashima, D. et al. Protein expression profiling identifies maspin and stathmin as potential biomarkers of adenoid cystic carcinoma of the salivary glands. Int. J. Cancer 118, 704–713 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Nishimori, T. et al. Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin. Proteomics 6, 1011–1018 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Seike, M. et al. Proteomic signatures for histological types of lung cancer. Proteomics 5, 2939–2948 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Somiari, R.I., Somiari, S., Russell, S. & Shriver, C.D. Proteomics of breast carcinoma. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 815, 215–225 (2005).

    Article  CAS  Google Scholar 

  43. Seike, M. et al. Proteomic signature of human cancer cells. Proteomics 4, 2776–2788 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Somiari, R.I. et al. High-throughput proteomic analysis of human infiltrating ductal carcinoma of the breast. Proteomics 3, 1863–1873 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Chan, H.L. et al. Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis. Proteomics 5, 2908–2926 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Alfonso, P. et al. Proteomic expression analysis of colorectal cancer by two-dimensional differential gel electrophoresis. Proteomics 5, 2602–2611 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Canals, F., Colome, N., Ferrer, C., Plaza-Calonge Mdel, C. & Rodriguez-Manzaneque, J.C. Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis. Proteomics 6 (suppl. 1): S28–S35 (2006).

    Article  PubMed  Google Scholar 

  48. Yu, K.H., Rustgi, A.K. & Blair, I.A. Characterization of proteins in human pancreatic cancer serum using differential gel electrophoresis and tandem mass spectrometry. J. Proteome Res. 4, 1742–1751 (2005).

    Article  CAS  PubMed  Google Scholar 

  49. Ornstein, D.K. & Petricoin, E.F., III Proteomics to diagnose human tumors and provide prognostic information. Oncology (Williston Park) 18, 521–529 discussion 529–532 (2004).

    Google Scholar 

  50. Alm, H. et al. Proteomic evaluation of neonatal exposure to 2,2,4,4,5-pentabromodiphenyl ether. Environ. Health Perspect. 114, 254–259 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. Roelens, S.A. et al. Neurotoxicity of polychlorinated biphenyls (PCBs) by disturbance of thyroid hormone-regulated genes. Ann. NY Acad. Sci. 1040, 454–456 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. Kleno, T.G. et al. Mechanisms of hydrazine toxicity in rat liver investigated by proteomics and multivariate data analysis. Proteomics 4, 868–880 (2004).

    Article  CAS  PubMed  Google Scholar 

  53. Ruepp, S.U., Tonge, R.P., Shaw, J., Wallis, N. & Pognan, F. Genomics and proteomics analysis of acetaminophen toxicity in mouse liver. Toxicol. Sci. 65, 135–150 (2002).

    Article  CAS  PubMed  Google Scholar 

  54. Tian, Q. et al. Integrated genomic and proteomic analyses of gene expression in mammalian cells. Mol. Cell Proteomics 3, 960–969 (2004).

    Article  CAS  PubMed  Google Scholar 

  55. Dowsey, A.W. et al. Examination of 2-DE in the Human Proteome Organisation Brain Proteome Project pilot studies with the new RAIN gel matching technique. Proteomics 6, 5030–5047 (2006).

    Article  CAS  PubMed  Google Scholar 

  56. Frohlich, T. et al. Analysis of the HUPO Brain proteome reference samples using 2-D DIGE and 2-D LC-MS/MS. Proteomics 6, 4950–4966 (2006).

    Article  PubMed  CAS  Google Scholar 

  57. Focking, M. et al. 2-D DIGE as a quantitative tool for investigating the HUPO Brain Proteome Project mouse series. Proteomics 6, 4914–4931 (2006).

    Article  CAS  PubMed  Google Scholar 

  58. Yan, J.X. et al. Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli. Proteomics 2, 1682–1698 (2002).

    Article  CAS  PubMed  Google Scholar 

  59. Gade, D., Thiermann, J., Markowsky, D. & Rabus, R. Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1. J. Mol. Microbiol. Biotechnol. 5, 240–251 (2003).

    Article  CAS  PubMed  Google Scholar 

  60. Corbett, M. et al. Identification of a new quorum-sensing-controlled virulence factor in Erwinia carotovora subsp. atroseptica secreted via the type II targeting pathway. Mol. Plant Microbe Interact. 18, 334–342 (2005).

    Article  CAS  PubMed  Google Scholar 

  61. Lopez-Campistrous, A. et al. Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth. Mol. Cell Proteomics 4, 1205–1209 (2005).

    Article  CAS  PubMed  Google Scholar 

  62. Gade, D., Gobom, J. & Rabus, R. Proteomic analysis of carbohydrate catabolism and regulation in the marine bacterium Rhodopirellula baltica. Proteomics 5, 3672–3683 (2005).

    Article  CAS  PubMed  Google Scholar 

  63. Rathsam, C. et al. Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms. J. Proteome Res. 4, 2161–2173 (2005).

    Article  CAS  PubMed  Google Scholar 

  64. VerBerkmoes, N.C. et al. Integrating “top-down” and “bottom-up” mass spectrometric approaches for proteomic analysis of Shewanella oneidensis. J. Proteome Res. 1, 239–252 (2002).

    Article  CAS  PubMed  Google Scholar 

  65. Busler, V.J. et al. Protein–protein interactions among Helicobacter pylori cag proteins. J. Bacteriol. 188, 4787–4800 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Vipond, C. et al. Proteomic analysis of a meningococcal outer membrane vesicle vaccine prepared from the group B strain NZ98/254. Proteomics 6, 3400–3413 (2006).

    Article  CAS  PubMed  Google Scholar 

  67. Slabas, A.R., Suzuki, I., Murata, N., Simon, W.J. & Hall, J.J. Proteomic analysis of the heat shock response in Synechocystis PCC6803 and a thermally tolerant knockout strain lacking the histidine kinase 34 gene. Proteomics 6, 845–864 (2006).

    Article  CAS  PubMed  Google Scholar 

  68. Tannu, N.S., Sanchez-Brambila, G., Kirby, P. & Andacht, T.M. Effect of staining reagent on peptide mass fingerprinting from in-gel trypsin digestions: a comparison of SyproRubytrade mark and DeepPurpletrade mark. Electrophoresis 27, 3136–3143 (2006).

    Article  CAS  PubMed  Google Scholar 

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  1. Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, 27157, North Carolina, USA

    Nilesh S Tannu & Scott E Hemby

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Tannu, N., Hemby, S. Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling. Nat Protoc 1, 1732–1742 (2006). https://doi.org/10.1038/nprot.2006.256

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