Advertisement

2D Gel Electrophoresis and Mass Spectrometry Identification and Analysis of Proteins

  • Paula MeleadyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 784)

Abstract

Analysis of the protein expression patterns in clinical samples and cells by proteomic technologies offers opportunities to discover potentially new biomarkers for early detection and diagnosis of disease. One of the most widely used techniques to study the proteome of a biological system is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In particular, a modified version of 2D-PAGE, two-dimensional difference gel electrophoresis (2D-DIGE), which uses differential labelling of protein samples with up to three fluorescent tags, offers greater sensitivity and reproducibility over conventional 2D-PAGE. In this chapter, we will introduce methods for the analysis of whole cell lysates from human cancer cell lines using 2D-DIGE and identification of differentially expressed proteins using liquid chromatography mass spectrometry, i.e. LC–MS/MS.

Key words

Two-dimensional gel electrophoresis Two-dimensional difference gel electrophoresis Mass spectrometry Protein expression profiling Proteomics 

References

  1. 1.
    Hanash, S. (2003) Disease proteomics. Nature 422, 226–232.PubMedCrossRefGoogle Scholar
  2. 2.
    Görg, A., Weiss, W., and Dunn, W.J. (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685.PubMedCrossRefGoogle Scholar
  3. 3.
    Görg, A., Drews, O., Lück, C., Weiland, F., and Weiss, W. (2009) 2-DE with IPGs. Electrophoresis. 30, Suppl 1:S122–32.Google Scholar
  4. 4.
    Smith, R. (2009) Two-Dimensional Electro­phoresis: An Overview. Methods Mol. Biol. 519, 1–16.PubMedCrossRefGoogle Scholar
  5. 5.
    Unlü, M., Morgan, M.E., and Minden, J.S. (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077.PubMedCrossRefGoogle Scholar
  6. 6.
    Alban, A., David, S.O., Bjorkesten, L., Andersson, C., Sloge, E., Lewis, S., and Currie, I. (2003) A novel experimental design for comparative two-dimensional analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3, 36–44.PubMedCrossRefGoogle Scholar
  7. 7.
    Mann, M., Hendrickson, R.C., and Pandey, A. (2001) Analysis of proteins and proteomes by mass spectrometry. Annu. Rev. Biochem. 70, 437–473.PubMedCrossRefGoogle Scholar
  8. 8.
    Corona, G., De Lorenzo, E., Elia, C., Simula, M.P., Avellini, C., Baccarani, U., Lupo, F., Tiribelli, C., Colombatti, A., and Toffoli, G. (2010) Differential proteomic analysis of hepatocellular carcinoma. Int. J. Oncol. 36, 93–9.PubMedGoogle Scholar
  9. 9.
    Dowling, P., O’ Driscoll, L., Meleady, P., Henry, M., Roy, S., Ballot, J., Moriarty, M., Crown, J., and Clynes, M. (2007) Two-dimensional difference gel electrophoresis of the lung squamous cell carcinoma versus normal sera demonstrates consistent alterations in the levels of 10 specific proteins. Electrophoresis 28, 4302–10.PubMedCrossRefGoogle Scholar
  10. 10.
    Dowling, P., Wormald, R., Meleady, P., Henry, M., Curran, A., and Clynes, M. (2008) Analysis of the saliva proteome from patients with head and neck squamous cell carcinoma reveals differences in abundance levels of proteins associated with tumour progression and metastasis. J. Proteomics 71, 168–75.CrossRefGoogle Scholar
  11. 11.
    Keenan, J., Murphy, L., Henry, M., Meleady, P., and Clynes, M (2009) Proteomic analysis of multidrug-resistance mechanisms in adriamycin-resistant variants of DLKP, a squamous lung cancer cell line. Proteomics 9, 1556–66.PubMedCrossRefGoogle Scholar
  12. 12.
    Lau, T.Y., Power, K.A., Dijon, S., de Gardelle, I., McDonnell, S., Duffy, M., Pennington, S., and Gallagher, W.M. (2010) Prioritization of candidate protein biomarkers from an in vitro model system of breast tumor progression towards clinical verification. J. Proteome Res. 9, 1450–9. Google Scholar
  13. 13.
    MacCoss, M.J., Wu, C.C., and Yates, J.R. 3rd. (2002) Probability-based validation of protein identifications using a modified SEQUEST algorithm. Anal. Chem. 74, 5593–5599.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.National Institute for Cellular BiotechnologyDublin City UniversityDublinIreland

Personalised recommendations