Two-Dimensional SDS-PAGE Fractionation of Biological Samples for Biomarker Discovery

  • Thierry Rabilloud
  • Sarah Triboulet
Part of the Methods in Molecular Biology book series (MIMB, volume 1002)


Two-dimensional electrophoresis is still a very valuable tool in proteomics, due to its reproducibility and its ability to analyze complete proteins. However, due to its sensitivity to dynamic range issues, its most suitable use in the frame of biomarker discovery is not on very complex fluids such as plasma, but rather on more proximal, simpler fluids such as CSF, urine, or secretome samples. Here, we describe the complete workflow for the analysis of such dilute samples by two-dimensional electrophoresis, starting from sample concentration, then the two-dimensional electrophoresis step per se, ending with the protein detection by fluorescence.

Key words

2D-PAGE Fluorescence dyes Image analysis Sample preparation Secretome Biological fluids 


  1. 1.
    Lescuyer P, Hochstrasser D, Rabilloud T (2007) How shall we use the proteomics toolbox for biomarker discovery? J Proteome Res 6:3371–3376PubMedCrossRefGoogle Scholar
  2. 2.
    Teng PN, Bateman NW, Hood BL et al (2010) Advances in proximal fluid proteomics for disease biomarker discovery. J Proteome Res 9:6091–6100PubMedCrossRefGoogle Scholar
  3. 3.
    Choi YS, Choe LH, Lee KH (2010) Recent cerebrospinal fluid biomarker studies of Alzheimer’s disease. Expert Rev Proteomics 7:919–926PubMedCrossRefGoogle Scholar
  4. 4.
    Kroksveen AC, Opsahl JA, Aye TT et al (2011) Proteomics of human cerebrospinal fluid: discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics. J Proteomics 74:371–388PubMedCrossRefGoogle Scholar
  5. 5.
    Maurer MH (2010) Proteomics of brain extracellular fluid (ECF) and cerebrospinal fluid (CSF). Mass Spectrom Rev 29:17–28PubMedGoogle Scholar
  6. 6.
    Zanusso G, Fiorini M, Ferrari S et al (2011) Cerebrospinal fluid markers in sporadic Creutzfeldt-Jakob disease. Int J Mol Sci 12:6281–6292PubMedCrossRefGoogle Scholar
  7. 7.
    Zhang J (2007) Proteomics of human cerebrospinal fluid—the good, the bad, and the ugly. Proteomics Clin Appl 41:805–819CrossRefGoogle Scholar
  8. 8.
    Casado-Vela J, del Pulgar TG, Cebrian A et al (2011) Human urine proteomics: building a list of human urine cancer biomarkers. Expert Rev Proteomics 8:347–360PubMedCrossRefGoogle Scholar
  9. 9.
    Julian BA, Suzuki H, Suzuki Y et al (2009) Sources of urinary proteins and their analysis by urinary proteomics for the detection of biomarkers of disease. Proteomics Clin Appl 3:1029–1043PubMedCrossRefGoogle Scholar
  10. 10.
    Mischak H, Kolch W, Aivaliotis M et al (2010) Comprehensive human urine standards for comparability and standardization in clinical proteome analysis. Proteomics Clin Appl 4:464–478PubMedCrossRefGoogle Scholar
  11. 11.
    Schaaij-Visser TB, Proost N, Nagel R et al (2011) Secretome proteomics to identify indicators for lung cancer treatment response prediction and monitoring. J Thorac Oncol 6:S1011Google Scholar
  12. 12.
    Makridakis M, Roubelaids MG, Bitsika V et al (2010) Analysis of secreted proteins for the study of bladder cancer cell aggressiveness. J Proteome Res 9:3243–3259PubMedCrossRefGoogle Scholar
  13. 13.
    Makridakis M, Vlahou A (2010) Secretome proteomics for discovery of cancer biomarkers. J Proteomics 73:2291–2305PubMedCrossRefGoogle Scholar
  14. 14.
    Luo XY, Liu YS, Wang R et al (2011) A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer. J Proteomics 74:528–538PubMedCrossRefGoogle Scholar
  15. 15.
    Caccia D, Domingues LZ, Micciche F et al (2011) Secretome compartment is a valuable source of biomarkers for cancer-relevant pathways. J Proteome Res 10:4196–4207PubMedCrossRefGoogle Scholar
  16. 16.
    Sarkissian G, Fergelot P, Lamy PJ et al (2008) Identification of Pro-MMP-7 as a serum marker for renal cell carcinoma by use of proteomic analysis. Clin Chem 54:574–581PubMedCrossRefGoogle Scholar
  17. 17.
    Roessler M, Rollinger W, Mantovani-Endl L et al (2006) Identification of PSME3 as a novel serum tumor marker for colorectal cancer by combining two-dimensional polyacrylamide gel electrophoresis with a strictly mass spectrometry-based approach for data analysis. Mol Cell Proteomics 5:2092–2101PubMedCrossRefGoogle Scholar
  18. 18.
    Celis JE (2004) Gel-based proteomics: what does MCP expect? Mol Cell Proteomics 3:949PubMedGoogle Scholar
  19. 19.
    Rabilloud T, Chevallet M, Luche S et al (2011) Two-dimensional gel electrophoresis in proteomics: past, present and future. J Proteomics 73:2064–2077CrossRefGoogle Scholar
  20. 20.
    Rabilloud T (2009) Membrane proteins and proteomics: love is possible, but so difficult. Electrophoresis 30(Suppl 1):S174–S180PubMedCrossRefGoogle Scholar
  21. 21.
    Yi JZ, Liu ZX, Craft D et al (2008) Intrinsic peptidase activity causes a sequential multi-step reaction (SMSR) in digestion of human plasma peptides. J Proteome Res 7:5112–5118PubMedCrossRefGoogle Scholar
  22. 22.
    Hoofnagle AN (2010) Peptide lost and found: internal standards and the mass spectrometric quantification of peptides. Clin Chem 56:1515–1517PubMedCrossRefGoogle Scholar
  23. 23.
    Bystrom CE, Salameh W, Reitz R et al (2010) Plasma renin activity by LC-MS/MS: development of a prototypical clinical assay reveals a subpopulation of human plasma samples with substantial peptidase activity. Clin Chem 56:1561–1569PubMedCrossRefGoogle Scholar
  24. 24.
    Hsich G, Kinney K, Gibbs CJ et al (1996) The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies. N Engl J Med 335:924–930PubMedCrossRefGoogle Scholar
  25. 25.
    Ladogana A, Sanchez-Juan P, Mitrova E et al (2009) Cerebrospinal fluid biomarkers in human genetic transmissible spongiform encephalopathies. J Neurol 256:1620–1628PubMedCrossRefGoogle Scholar
  26. 26.
    Bartosik-Psujek H, Archelos JJ (2004) Tau protein and 14-3-3 are elevated in the cerebrospinal fluid of patients with multiple sclerosis and correlate with intrathecal synthesis of IgG. J Neurol 251:414–420PubMedCrossRefGoogle Scholar
  27. 27.
    Colucci M, Roccatagliata L, Capello E et al (2004) The 14-3-3 protein in multiple sclerosis: a marker of disease severity. Mult Scler 10:477–481PubMedCrossRefGoogle Scholar
  28. 28.
    Pitarch A, Nombela C, Gil C (2009) Proteomic profiling of serologic response to Candida albicans during host-commensal and host-pathogen interactions. Methods Mol Biol 470:369–411PubMedCrossRefGoogle Scholar
  29. 29.
    Pitarch A, Nombela C, Gil C (2011) Prediction of the clinical outcome in invasive candidiasis patients based on molecular fingerprints of five anti-Candida antibodies in serum. Mol Cell Proteomics 10(M110):004010PubMedGoogle Scholar
  30. 30.
    Maass S, Sievers S, Zuhlke D et al (2011) Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics. Anal Chem 83:2677–2684PubMedCrossRefGoogle Scholar
  31. 31.
    Aude-Garcia C, Collin-Faure V, Luche S et al (2011) Improvements and simplifications in in-gel fluorescent detection of proteins using ruthenium II tris-(bathophenanthroline disulfonate): the poor man’s fluorescent detection method. Proteomics 11:324–328PubMedCrossRefGoogle Scholar
  32. 32.
    Chevallet M, Diemer H, Van Dorssealer A et al (2007) Toward a better analysis of secreted proteins: the example of the myeloid cells secretome. Proteomics 7:1757–1770PubMedCrossRefGoogle Scholar
  33. 33.
    Luche S, Diemer H, Tastet C et al (2004) About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis. Proteomics 4:551–561PubMedCrossRefGoogle Scholar
  34. 34.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  35. 35.
    Tastet C, Lescuyer P, Diemer H et al (2003) A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins. Electrophoresis 24:1787–1794PubMedCrossRefGoogle Scholar
  36. 36.
    Dowell JA, Johnson JA, Li LJ (2009) Identification of astrocyte secreted proteins with a combination of shotgun proteomics and bioinformatics. J Proteome Res 8:4135–4143PubMedCrossRefGoogle Scholar
  37. 37.
    Sanchez JC, Hochstrasser D, Rabilloud T (1999) In-gel sample rehydration of immobilized pH gradient. Methods Mol Biol 112:221–225PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Thierry Rabilloud
    • 1
  • Sarah Triboulet
    • 2
  1. 1.Chemistry and Biology of Metals GrenobleCEA GrenobleGrenobleFrance
  2. 2.Université Joseph FourierGrenobleFrance

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