Abstract
The global analysis of the proteome is an important tool in cell biology. Comparative proteomic evaluations can identify and compare the composition, dynamics, and modifications between different samples. Comparing tissue proteomes under different conditions is crucial for advancing the biomedical field. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a sensitive and robust biochemical method that can compare multiple protein samples over a broad dynamic range on the same analytical gel and can be used to establish differentially expressed protein profiles between different sample groups. 2D-DIGE involves fluorescently labeling protein samples with CyDye flours, via a two-dye or a three-dye system, pre-separation by isoelectric point, and molecular weight. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization, thus enabling accurate high-resolution analysis of differences in protein abundance between samples. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of two-dye and three-dye DIGE minimal labeling.
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References
Meissner F, Mann M (2014) Quantitative shotgun proteomics: considerations for a high-quality workflow in immunology. Nat Immunol 15(2):112–117. https://doi.org/10.1038/ni.2781
Wilhelm M, Schlegl J, Hahne H et al (2014) Mass-spectrometry-based draft of the human proteome. Nature 509(7502):582–587. https://doi.org/10.1038/nature13319
Dubowitz V, Sewry CA, Oldfors A (2013) Muscle biopsy: a practical approach, 4th edn. Saunders Elsevier, Philadelphia
Holland A, Ohlendieck K (2015) Comparative profiling of sperm proteome. Proteomics 15(4):632–648. https://doi.org/10.1002/pmic.201400032
Jockusch H, Holland A, Staunton L, Schmitt-John T, Heimann P, Dowling P, Ohlendieck K (2014) Pathoproteomics of testicular tissue deficient in the GARP component VPS54: the wobbler mouse model of globozoospermia. Proteomics 14(7–8):839–852. https://doi.org/10.1002/pmic.201300189
Holland A, Ohlendieck K (2014) Proteomic identification of muscle-associated biomarkers of amyotrophic lateral sclerosis using the wobbler mouse model of primary motor neuropathy. J Integr OMICS 4(2):57–68. https://doi.org/10.5584/jiomics.v4i2.171
Liang J, Zheng Y, Zeng W, Chen L, Yang S, Du P, Wang Y, Yu X, Zhang X (2021) Comparison of proteomic profiles from the testicular tissue of males with impaired and normal spermatogenesis. Syst Biol Reprod Med 67(2):127–136. https://doi.org/10.1080/19396368.2020.1846822
**ong W, Ge H, Shen C, Li C, Zhang X, Tang L, Shen Y, Lu S, Zhang H, Wang Z (2022) PRSS37 deficiency leads to impaired energy metabolism in testis and sperm revealed by DIA-based quantitative proteomic analysis. Reprod Sci. https://doi.org/10.1007/s43032-022-00918-x. Epub ahead of print. PMID: 35471551.
Dowling P, Holland A, Ohlendieck K (2014) Mass spectrometry-based identification of muscle-associated and muscle derived proteomic biomarkers of dystrophinopathies. J Neuromuscul Dis 1(1):15–40. https://doi.org/10.3233/JND-140011
Rabilloud T, Lelong C (2011) Two-dimensional gel electrophoresis in proteomics: a tutorial. J Proteome 74(10):1829–18241. https://doi.org/10.1016/j.jprot.2011.05.040
Murphy S, Dowling P, Ohlendieck K (2016) Comparative skeletal muscle proteomics using two-dimensional gel electrophoresis. Proteomes 4(3):27. https://doi.org/10.3390/proteomes4030027
O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250(10):4007–4021
Rible H (1973) Historical and theoretical aspects of isoelectric focusing. Ann N Y Acad Sci 209:11–22
Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18(11):2071–2077. https://doi.org/10.1002/elps.1150181133
Arentz G, Weiland F, Oehler MK, Hoffmann P (2015) State of the art of 2D DIGE. Proteomics Clin Appl 9(3–4):277–288. https://doi.org/10.1002/prca.201400119
Minden JS (2012) DIGE: past and future. Methods Mol Biol 854:3–8. https://doi.org/10.1007/978-1-61779-573-2_1
Ohlendieck K (2018) Comparative DIGE proteomics. Methods Mol Biol 1664:17–24. https://doi.org/10.1007/978-1-4939-7268-5_2. PMID: 29019121.
Ohlendieck K (1664) Comparative 3-sample DIGE analysis of skeletal muscles. Methods Mol Biol 2018:93–108. https://doi.org/10.1007/978-1-4939-7268-5_9. PMID: 29019128.
Shaw J, Rowlinson R, Nickson J, Stone T, Sweet A, Williams K, Tongue R (2003) Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3(7):1181–1195. https://doi.org/10.1002/pmic.200300439
Tonge R, Shaw J, Middleton B, Rowlingson 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):377–396. https://doi.org/10.1002/1615-9861(200103)1:3,377:AID-PROT377>3.0.CO;2-6
Viswanathan S, Unlu M, Minden JS (2006) Two-dimensional difference gel electrophoresis. Nat Protoc 1(3):1351–1358. https://doi.org/10.1038/nprot.2006.234
Minden J (2007) Comparative proteomics and difference gel electrophoresis. BioTechniques 43(6):739–745
Karp NA, Lilley KS (2005) Maximising sensitivity for detecting changes in protein expression: experimental design using minimal CyDyes. Proteomics 5(12):3105–3115. https://doi.org/10.1002/pmic.200500083
Karp NA, McCormick PS, Russell MR, Lilley KS (2007) Experimental and statistical consideration to avoid false conclusions in proteomics studies using differential in-gel electrophoresis. Mol Cell Proteomics 6(8):1354–1364. https://doi.org/10.1074/mcp.M600274-MCP200
Holland A, Schmitt-John T, Dowling P, Meleady P, Henry M, Clynes M, Ohlendieck K (2014) Intricate effects of primary motor neuropathy on contractile proteins and metabolic muscle enzymes as revealed by label-free mass spectrometry. Biosci Rep 34(4):331–343. https://doi.org/10.1042/BSR20140029
Holland A, Ohlendieck K (2014) Comparative proteomics for studying muscular dystrophy: intrinsic biological and analytical issues associated with the systemic utilization of tissue specimens. J Proteom Bioinform S10:002. https://doi.org/10.4172/jbs.S10-002
Beckett P (2012) The basics of 2D DIGE. Difference gel electrophoresis (DIGE): methods and protocols. Methods Mol Biol 854:9–18. https://doi.org/10.1007/978-1-61779-573-2_2
Karpievitch YV, Polpitiya AD, Anderson GA, Smith RD, Dabney AR (2010) Liquid chromatography mass spectrometry-based proteomics: biological and technological aspects. Ann Appl Stat 4(4):1797–1823. https://doi.org/10.1214/10-AOAS341. PMID: 21593992; PMCID: PMC3095207
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Holland, A. (2023). Two-Dye Versus Three-Dye DIGE for Comparative Testis Tissue Proteomic Analysis. In: Ohlendieck, K. (eds) Difference Gel Electrophoresis. Methods in Molecular Biology, vol 2596. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2831-7_18
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