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DIGE-Based Phosphoproteomic Analysis

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Difference Gel Electrophoresis

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

Abstract

Here, we describe a detailed step-by-step protocol for the detection of phosphoproteins in two-dimensional difference gel electrophoresis (2D-DIGE) gels. A standard 2D-DIGE protocol is combined with subsequent post-staining with phosphospecific fluorescent dye. The combination of these two methods complements 2D-DIGE-based proteome profiling by fluorescence detection of phosphoproteins in the same gel providing additional possibility for sensitive and accurate quantification of the differentially regulated phosphoproteins in biological samples.

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References

  1. Görg A, Weiss W, Dunn MJ (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4(12):3665–3685. https://doi.org/10.1002/pmic.200401031

    Article  PubMed  CAS  Google Scholar 

  2. Patton WF, Beechem JM (2002) Rainbow’s end: the quest for multiplexed fluorescence quantitative analysis in proteomics. Curr Opin Chem Biol 6(1):63–69

    Article  PubMed  CAS  Google Scholar 

  3. Ünlü 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

    Article  PubMed  Google Scholar 

  4. Mackintosh JA, Choi HY, Bae SH, Veal DA, Bell PJ, Ferrari BC, Van Dyk DD, Verrills NM, Paik YK, Karuso P (2003) A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis. Proteomics 3(12):2273–2288. https://doi.org/10.1002/pmic.200300578

    Article  PubMed  CAS  Google Scholar 

  5. Berggren KN, Schulenberg B, Lopez MF, Steinberg TH, Bogdanova A, Smejkal G, Wang A, Patton WF (2002) An improved formulation of SYPRO Ruby protein gel stain: comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation. Proteomics 2(5):486–498. https://doi.org/10.1002/1615-9861(200205)2:5<486::AID-PROT486>3.0.CO;2-X

    Article  PubMed  CAS  Google Scholar 

  6. Rabilloud T, Strub JM, Luche S, van Dorsselaer A, Lunardi J (2001) A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels. Proteomics 1(5):699–704. https://doi.org/10.1002/1615-9861(200104)1:5<699::AID-PROT699>3.0.CO;2-C

    Article  PubMed  CAS  Google Scholar 

  7. Paulo JA, Schweppe DK (2021) Advances in quantitative high-throughput phosphoproteomics with sample multiplexing. Proteomics 21(9):e2000140. https://doi.org/10.1002/pmic.202000140

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Low TY, Mohtar MA, Lee PY, Omar N, Zhou H, Ye M (2021) Widening the bottleneck of phosphoproteomics: evolving strategies for phosphopeptide enrichment. Mass Spectrom Rev 40(4):309–333. https://doi.org/10.1002/mas.21636

    Article  PubMed  CAS  Google Scholar 

  9. Mantini G, Pham TV, Piersma SR, Jimenez CR (2021) Computational analysis of phosphoproteomics data in multi-omics cancer studies. Proteomics 21(3–4):e1900312. https://doi.org/10.1002/pmic.201900312

    Article  PubMed  CAS  Google Scholar 

  10. Urban J (2022) A review on recent trends in the phosphoproteomics workflow. From sample preparation to data analysis. Anal Chim Acta 1199:338857. https://doi.org/10.1016/j.aca.2021.338857

    Article  PubMed  CAS  Google Scholar 

  11. Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J, Beechem JM, Haugland RP, Patton WF (2003) Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics 3(7):1128–1144. https://doi.org/10.1002/pmic.200300434

    Article  PubMed  CAS  Google Scholar 

  12. Stasyk T, Morandell S, Bakry R, Feuerstein I, Huck CW, Stecher G, Bonn GK, Huber LA (2005) Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining. Electrophoresis 26(14):2850–2854. https://doi.org/10.1002/elps.200500026

    Article  PubMed  CAS  Google Scholar 

  13. de Araùjo ME, Huber LA, Stasyk T (2008) Isolation of endocitic organelles by density gradient centrifugation. Methods Mol Biol 424:317–331. https://doi.org/10.1007/978-1-60327-064-9_25

    Article  PubMed  Google Scholar 

  14. Stasyk T, Schiefermeier N, Skvortsov S, Zwierzina H, Peränen J, Bonn GK, Huber LA (2007) Identification of endosomal epidermal growth factor receptor signaling targets by functional organelle proteomics. Mol Cell Proteomics 6(5):908–922. https://doi.org/10.1074/mcp.M600463-MCP200

    Article  PubMed  CAS  Google Scholar 

  15. Orsatti L, Forte E, Tomei L, Caterino M, Pessi A, Talamo F (2009) 2-D Difference in gel electrophoresis combined with Pro-Q Diamond staining: a successful approach for the identification of kinase/phosphatase targets. Electrophoresis 30(14):2469–2476. https://doi.org/10.1002/elps.200800780

    Article  PubMed  CAS  Google Scholar 

  16. Mattei B, Spinelli F, Pontiggia D, De Lorenzo G (2016) Comprehensive analysis of the membrane phosphoproteome regulated by oligogalacturonides in Arabidopsis thaliana. Front Plant Sci 7:1107. https://doi.org/10.3389/fpls.2016.01107

    Article  PubMed  PubMed Central  Google Scholar 

  17. Lamanda A, Zahn A, Roder D, Langen H (2004) Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal-tobackground ratio and improved baseline resolution. Proteomics 4(3):599–608. https://doi.org/10.1002/pmic.200300587

    Article  PubMed  CAS  Google Scholar 

  18. Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138(1):141–143

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Taras Stasyk .

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Stasyk, T., Huber, L.A. (2023). DIGE-Based Phosphoproteomic 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_7

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  • DOI: https://doi.org/10.1007/978-1-0716-2831-7_7

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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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