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DIGE Analysis Software and Protein Identification Approaches

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Difference Gel Electrophoresis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2596))

Abstract

Two-dimensional difference gel electrophoresis (2D-DIGE) is a high-resolution protein separation technique, with the excellent dynamic range obtained by fluorescent tag labeling of protein samples. Scanned images of 2D-DIGE gels show thousands of protein spots, each spot representing a single or a group of protein isoforms. By using commercially available software, each protein spot is defined by an outline, which is digitized and correlated with the quantity of proteins present in each spot. Software packages include DeCyder, SameSpots, and Dymension 3. In addition, proteins of interest can be excised from post-stained gels and identified with conventional mass spectrometric techniques. High-throughput mass spectrometry is performed using sophisticated instrumentation, including matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), MALDI-TOF/TOF, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Tandem MS (MALDI-TOF/TOF or LC-MS/MS) analyzes fragmented peptides, resulting in amino acid sequence information, which is especially useful when protein spots are low abundant or where a mixture of proteins is present.

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References

  1. Dowling P, O'Driscoll L, O'Sullivan F, Dowd A, Henry M, Jeppesen PB, Meleady P, Clynes M (2006) Proteomic screening of glucose-responsive and glucose non-responsive MIN-6 beta cells reveals differential expression of proteins involved in protein folding, secretion and oxidative stress. Proteomics 6:6578–6587

    Article  PubMed  CAS  Google Scholar 

  2. Gupta S, Ghulmiyyah J, Sharma R, Halabi J, Agarwal A (2014) Power of proteomics in linking oxidative stress and female infertility. Biomed Res Int 2014:916212

    Article  PubMed  PubMed Central  Google Scholar 

  3. Kang Y, Techanukul T, Mantalaris A, Nagy JM (2009) Comparison of three commercially available DIGE analysis software packages: minimal user intervention in gel-based proteomics. J Proteome Res 8:1077–1084

    Article  PubMed  CAS  Google Scholar 

  4. Malm C, Hadrevi J, Bergström SA, Pedrosa-Domellöf F, Antti H, Svensson M, Frängsmyr L (2008) Evaluation of 2-D DIGE for skeletal muscle: protocol and repeatability. Scand J Clin Lab Invest 68:793–800

    Article  PubMed  CAS  Google Scholar 

  5. Doran P, Martin G, Dowling P, Jockusch H, Ohlendieck K (2006) Proteome analysis of the dystrophin-deficient MDX diaphragm reveals a drastic increase in the heat shock protein cvHSP. Proteomics 6:4610–4621

    Article  PubMed  CAS  Google Scholar 

  6. Missous G, Thammavongs B, Dieuleveux V, Houssin M, Henry J, Panoff JM (2012) Proteomic expression of microfungal ripening starter Geotrichum candidum submitted to cold stress is strain-dependent: studies using 2d-dige technology and samespots software analysis. Cryo Letters 33:289–298

    PubMed  CAS  Google Scholar 

  7. Donoghue P, Staunton L, Mullen E, Manning G, Ohlendieck K (2010) DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue. J Proteome 73:1441–1453

    Article  CAS  Google Scholar 

  8. Soares AJ, Santos MF, Trugilho MR, Neves-Ferreira AG, Perales J, Domont GB (2009) Differential proteomics of the plasma of individuals with sepsis caused by Acinetobacter baumannii. J Proteome 73:267–278

    Article  CAS  Google Scholar 

  9. Doran P, Wilton SD, Fletcher S, Ohlendieck K (2009) Proteomic profiling of antisense-induced exon skipping reveals reversal of pathobiochemical abnormalities in dystrophic mdx diaphragm. Proteomics 9:671–685

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Ferrín G, Rodríguez-Perálvarez M, Aguilar-Melero P, Ranchal I, Llamoza C, Linares CI, González-Rubio S, Muntané J, Briceño J, López-Cillero P, Montero-Álvarez JL, de la Mata M (2015) Plasma protein biomarkers of hepatocellular carcinoma in HCV-infected alcoholic patients with cirrhosis. PLoS One 10:e0118527

    Article  PubMed  PubMed Central  Google Scholar 

  11. Doran P, O'Connell K, Gannon J, Kavanagh M, Ohlendieck K (2008) Opposite pathobiochemical fate of pyruvate kinase and adenylate kinase in aged rat skeletal muscle as revealed by proteomic DIGE analysis. Proteomics 8:364–377

    Article  PubMed  CAS  Google Scholar 

  12. Weng CY, Wang CE, Xie WB, Xu SY, Wang YJ, Zheng YG (2021) Comparative proteome analysis of Actinoplanes utahensis grown on various saccharides based on 2D-DIGE and MALDI-TOF/TOF-MS. J Proteome 239:104193

    Article  CAS  Google Scholar 

  13. Davalieva K, Kiprijanovska S, Komina S, Petrusevska G, Zografska NC, Polenakovic M (2015) Proteomics analysis of urine reveals acute phase response proteins as candidate diagnostic biomarkers for prostate cancer. Proteome Sci 13:2

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Eustace AJ, Dowling P, Henry M, Doolan P, Meleady P, Clynes M, Crown J, O'Donovan N (2011) 2D-DIGE analysis of phospho-enriched fractions from dasatinib-treated melanoma cell lines. J Proteome 74:490–501

    Article  CAS  Google Scholar 

  15. Korte J, Fröhlich T, Kohn M, Kaspers B, Arnold GJ, Härtle S (2013) 2D DIGE analysis of the bursa of Fabricius reveals characteristic proteome profiles for different stages of chicken B-cell development. Proteomics 13:119–133

    Article  PubMed  CAS  Google Scholar 

  16. Davalieva K, Kostovska IM, Kiprijanovska S, Markoska K, Kubelka-Sabit K, Filipovski V, Stavridis S, Stankov O, Komina S, Petrusevska G, Polenakovic M (2015) Proteomics analysis of malignant and benign prostate tissue by 2D DIGE/MS reveals new insights into proteins involved in prostate cancer. Prostate 75:1586–1600

    Article  PubMed  CAS  Google Scholar 

  17. Ciereszko A, Dietrich MA, Słowińska M, Nynca J, Ciborowski M, Kaczmarek MM, Myszczyński K, Kiśluk J, Majewska A, Michalska-Falkowska A, Kodzik N, Reszeć J, Sierko E, Nikliński J (2022) Application of two-dimensional difference gel electrophoresis to identify protein changes between center, margin, and adjacent non-tumor tissues obtained from non-small-cell lung cancer with adenocarcinoma or squamous cell carcinoma subtype. PLoS One 17:e0268073

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Karp NA, Feret R, Rubtsov DV, Lilley KS (2008) Comparison of DIGE and post-stained gel electrophoresis with both traditional and SameSpots analysis for quantitative proteomics. Proteomics 8:948–960

    Article  PubMed  CAS  Google Scholar 

  19. Tokutomi Y, Araki N, Kataoka K, Yamamoto E, Kim-Mitsuyama S (2007) Oxidation of Prx2 and phosphorylation of GRP58 by angiotensin II in human coronary smooth muscle cells identified by 2D-DIGE analysis. Biochem Biophys Res Commun 364:822–830

    Article  PubMed  CAS  Google Scholar 

  20. Tewari AK, Popova-Butler A, El-Mahdy MA, Zweier JL (2011) Identification of differentially expressed proteins in blood plasma of control and cigarette smoke-exposed mice by 2-D DIGE/MS. Proteomics 11:2051–2062

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Sivagnanam A, Shyamsundar V, Kesavan P, Krishnamurthy A, Thangaraj SV, Venugopal DC, Kasirajan H, Ramani P, Sarma VR, Ramshankar V (2022) 2D-DIGE-based proteomic profiling with validations identifies vimentin as a secretory biomarker useful for early detection and poor prognosis in oral cancers. J Oncol 2022:4215097

    Article  PubMed  PubMed Central  Google Scholar 

  22. Ignjatovic V, Lai C, Summerhayes R, Mathesius U, Tawfilis S, Perugini MA, Monagle P (2011) Age-related differences in plasma proteins: how plasma proteins change from neonates to adults. PLoS One 6:e17213

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Zurawel A, Moore EE, Peltz ED, Jordan JR, Damle S, Dzieciatkowska M, Banerjee A, Hansen KC (2010) Proteomic profiling of the mesenteric lymph after hemorrhagic shock: differential gel electrophoresis and mass spectrometry analysis. Clin Proteomics 8:1

    Article  PubMed  PubMed Central  Google Scholar 

  24. Grzelak S, Stachyra A, Moskwa B, Bień-Kalinowska J (2021) Exploiting the potential of 2D DIGE and 2DE immunoblotting for comparative analysis of crude extract of Trichinella britovi and Trichinella spiralis muscle larvae proteomes. Vet Parasitol 289:109323

    Article  PubMed  CAS  Google Scholar 

  25. Bailey MJ, Shield-Artin KL, Oliva K, Ayhan M, Reisman S, Rice GE (2013) Stage-specific analysis of plasma protein profiles in ovarian cancer: difference in-gel electrophoresis analysis of pooled clinical samples. J Carcinog 12:10

    Article  PubMed  PubMed Central  Google Scholar 

  26. Ribeiro DM, Planchon S, Leclercq CC, Dentinho MTP, Bessa RJB, Santos-Silva J, Paulos K, Jerónimo E, Renaut J, Almeida AM (2020) The effects of improving low dietary protein utilization on the proteome of lamb tissues. J Proteome 223:103798

    Article  CAS  Google Scholar 

  27. Vélez P, Izquierdo I, Rosa I, García Á (2015) A 2D-DIGE-based proteomic analysis reveals differences in the platelet releasate composition when comparing thrombin and collagen stimulations. Sci Rep 5:8198

    Article  PubMed  PubMed Central  Google Scholar 

  28. Kendall RT, Renaud L, Baatz JE, Malaab M, Nguyen XX, Feghali-Bostwick CA (2021) Systemic sclerosis biomarkers detection in the secretome of TGFβ1-activated primary human lung fibroblasts. J Proteome 242:104243

    Article  CAS  Google Scholar 

  29. Bouvet M, Turkieh A, Acosta-Martin AE, Chwastyniak M, Beseme O, Amouyel P, Pinet F (2014) Proteomic profiling of macrophages by 2D electrophoresis. J Vis Exp:e52219

    Google Scholar 

  30. Heywood WE, Madgett TE, Wang D, Wallington A, Hogg J, Mills K, Avent ND (2011) 2D DIGE analysis of maternal plasma for potential biomarkers of Down Syndrome. Proteome Sci 9:56

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Choudhary MK, Nomura Y, Shi H, Nakagami H, Somers DE (2016) Circadian profiling of the arabidopsis proteome using 2D-DIGE. Front Plant Sci 7:1007

    Article  PubMed  PubMed Central  Google Scholar 

  32. Jiang YJ, Sun Q, Fang XS, Wang X (2009) Comparative mitochondrial proteomic analysis of Rji cells exposed to adriamycin. Mol Med 15:173–182

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Pressey JG, Pressey CS, Robinson G, Herring R, Wilson L, Kelly DR, Kim H (2011) 2D-difference gel electrophoretic proteomic analysis of a cell culture model of alveolar rhabdomyosarcoma. J Proteome Res 10:624–636

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Ramadoss J, Magness RR (2011) 2-D DIGE uterine endothelial proteomic profile for maternal chronic binge-like alcohol exposure. J Proteome 74:2986–2994

    Article  CAS  Google Scholar 

  35. Kultima K, Scholz B, Alm H, Sköld K, Svensson M, Crossman AR, Bezard E, Andrén PE, Lönnstedt I (2006) Normalization and expression changes in predefined sets of proteins using 2D gel electrophoresis: a proteomic study of L-DOPA induced dyskinesia in an animal model of Parkinson's disease using DIGE. BMC Bioinf 7:475

    Article  Google Scholar 

  36. Penque D (2009) Two-dimensional gel electrophoresis and mass spectrometry for biomarker discovery. Proteomics Clin Appl 3:155–172

    Article  PubMed  CAS  Google Scholar 

  37. Dowsey AW, Morris JS, Gutstein HB, Yang GZ (2010) Informatics and statistics for analyzing 2-d gel electrophoresis images. Methods Mol Biol 604:239–255

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Della Corte A, Maugeri N, Pampuch A, Cerletti C, de Gaetano G, Rotilio D (2008) Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome. Platelets 19:43–50

    Article  PubMed  CAS  Google Scholar 

  39. Chen J, Wu W, Chen L, Zhou H, Yang R, Hu L, Zhao Y (2013) Profiling the potential tumor markers of pancreatic ductal adenocarcinoma using 2D-DIGE and MALDI-TOF-MS: up-regulation of complement C3 and alpha-2-HS-glycoprotein. Pancreatology 13:290–297

    Article  PubMed  CAS  Google Scholar 

  40. Sikulu MT, Monkman J, Dave KA, Hastie ML, Dale PE, Kitching RL, Killeen GF, Kay BH, Gorman JJ, Hugo LE (2015) Mass spectrometry identification of age-associated proteins from the malaria mosquitoes Anopheles gambiae s.s. and Anopheles stephensi. Data Brief 4:461–467

    Article  PubMed  PubMed Central  Google Scholar 

  41. Lu J, Zhou Z, Zheng J, Zhang Z, Lu R, Liu H, Shi H, Tu Z (2015) 2D-DIGE and MALDI TOF/TOF MS analysis reveal that small GTPase signaling pathways may play an important role in cadmium-induced colon cell malignant transformation. Toxicol Appl Pharmacol 288:106–113

    Article  PubMed  CAS  Google Scholar 

  42. Weeks ME (2010) Urinary proteome profiling using 2D-DIGE and LC-MS/MS. Methods Mol Biol 658:293–309

    Article  PubMed  CAS  Google Scholar 

  43. Sinclair J, Metodieva G, Dafou D, Gayther SA, Timms JF (2011) Profiling signatures of ovarian cancer tumour suppression using 2D-DIGE and 2D-LC-MS/MS with tandem mass tagging. J Proteome 74:451–465

    Article  CAS  Google Scholar 

  44. Hammer E, Bien S, Salazar MG, Steil L, Scharf C, Hildebrandt P, Schroeder HW, Kroemer HK, Völker U, Ritter CA (2010) Proteomic analysis of doxorubicin-induced changes in the proteome of HepG2cells combining 2-D DIGE and LC-MS/MS approaches. Proteomics 10:99–114

    Article  PubMed  CAS  Google Scholar 

  45. Burniston JG, Kenyani J, Gray D, Guadagnin E, Jarman IH, Cobley JN, Cuthbertson DJ, Chen YW, Wastling JM, Lisboa PJ, Koch LG, Britton SL (2014) Conditional independence mapping of DIGE data reveals PDIA3 protein species as key nodes associated with muscle aerobic capacity. J Proteome 106:230–245

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biology, Maynooth University, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland

    Paul Dowling

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  1. Paul Dowling
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Correspondence to Paul Dowling .

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  1. Maynooth University, MAYNOOTH, Ireland

    Kay Ohlendieck

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Dowling, P. (2023). DIGE Analysis Software and Protein Identification Approaches. 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_3

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  • DOI: https://doi.org/10.1007/978-1-0716-2831-7_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2830-0

  • Online ISBN: 978-1-0716-2831-7

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