Protein Mass Spectrometry Applications on FFPE Tissue Sections

  • Carol B. FowlerEmail author
  • Timothy J. O’Leary
  • Jeffrey T. Mason
Part of the Methods in Molecular Biology book series (MIMB, volume 724)


Formalin-fixed, paraffin-embedded (FFPE) tissue archives and their associated diagnostic records represent an invaluable source of proteomic information on diseases where the patient outcomes are already known. Over the last few years, advances in methodology have made it possible to recover peptides from FFPE tissues that yield a reasonable representation of the proteins recovered from identical fresh or frozen specimens. These new methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, have developed sufficiently to allow at least a qualitative analysis of the proteome of FFPE archival tissues. This chapter describes the approaches for performing proteomic analysis on FFPE tissues by liquid chromatography and mass spectrometry.

Key words

Formalin-fixed paraffin-embedded Proteolytic digestion Proteomics Liquid chromatography Tandem mass spectrometry Protein extraction Gel electrophoresis Antigen retrieval Histology 


  1. 1.
    Blonder, J. and Veenstra, T. D. (2009) Clinical proteomic applications of formalin-fixed paraffin-embedded tissues. Clin Lab Med. 29, 101–13.PubMedCrossRefGoogle Scholar
  2. 2.
    Wilkinson, E. J. and Hendricks, J. B. (1995) Role of the pathologist in biomarker studies. J Cell Biochem Suppl. 23, 10–8.PubMedCrossRefGoogle Scholar
  3. 3.
    Patel, V., Hood, B. L., Molinolo, A. A., Lee, N. H., Conrads, T. P., Braisted, J. C., et al. (2008) Proteomic analysis of laser-captured paraffin-embedded tissues: a molecular portrait of head and neck cancer progression. Clin Cancer Res. 14, 1002–14.PubMedCrossRefGoogle Scholar
  4. 4.
    Negishi, A., Masuda, M., Ono, M., Honda, K., Shitashige, M., Satow, R., et al. (2009) Quantitative proteomics using formalin-fixed paraffin-embedded tissues of oral squamous cell carcinoma. Cancer Sci. 100, 1605–11.PubMedCrossRefGoogle Scholar
  5. 5.
    Jain, M. R., Liu, T., Hu, J., Darfler, M., Fitzhugh, V., Rinaggio, J., et al. (2008) Quantitative proteomic analysis of formalin fixed paraffin embedded oral HPV lesions from HIV patients. Open Proteomics J. 1, 40–5.PubMedCrossRefGoogle Scholar
  6. 6.
    Hood, B. L., Darfler, M. M., Guiel, T. G., Furusato, B., Lucas, D. A., Ringeisen, B. R., et al. (2005) Proteomic analysis of formalin-fixed prostate cancer tissue. Mol Cell Proteomics 4, 1741–53.PubMedCrossRefGoogle Scholar
  7. 7.
    Prieto, D. A., Hood, B. L., Darfler, M. M., Guiel, T. G., Lucas, D. A., Conrads, T. P., et al. (2005) Liquid tissue: proteomic profiling of formalin-fixed tissues. Biotechniques 38(Suppl), 32–5.CrossRefGoogle Scholar
  8. 8.
    Shi, S.-R., Liu, C., Balgley, B. M., Lee, C., and Taylor, C. R. (2006) Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry. J Histochem Cytochem. 54, 739–43.PubMedCrossRefGoogle Scholar
  9. 9.
    Guo, T., Wang, W., Rudnick, P. A., Song, T., Li, J., Zhuang, Z., et al. (2007) Proteome analysis of microdissected formalin-fixed and paraffin-embedded tissue specimens. J Histochem Cytochem. 55, 763–72.PubMedCrossRefGoogle Scholar
  10. 10.
    Xu, H., Yang, L., Wang, W., Shi, S. R., Liu, C., Liu, Y., et al. (2008) Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues. J Proteome Res. 7, 1098–108.PubMedCrossRefGoogle Scholar
  11. 11.
    Palmer-Toy, D. E., Krastins, B., Sarracino, D. A., Nadol, J. B., Jr., and Merchant, S. N. (2005) Efficient method for the proteomic analysis of fixed and embedded tissues. J Proteome Res. 4, 2404–11.PubMedCrossRefGoogle Scholar
  12. 12.
    Jiang, X., Jiang, X., Feng, S., Tian, R., Ye, M., and Zou, H. (2007) Development of efficient protein extraction methods for shotgun proteome analysis of formalin-fixed tissues. J Proteome Res. 6, 1038–47.PubMedCrossRefGoogle Scholar
  13. 13.
    Sprung, R. W., Jr., Brock, J. W., Tanksley, J. P., Li, M., Washington, M. K., Slebos, R. J., et al. (2009) Equivalence of protein inventories obtained from formalin-fixed paraffin-embedded and frozen tissue in multidimensional liquid chromatography-tandem mass spectrometry shotgun proteomic analysis. Mol Cell Proteomics 8, 1988–98.PubMedCrossRefGoogle Scholar
  14. 14.
    Fowler, C. B., Cunningham, R. E., O’Leary, T. J., and Mason, J. T. (2007) “Tissue surrogates” as a model for archival formalin-fixed paraffin-embedded tissues. Lab Invest. 87, 836–46.PubMedCrossRefGoogle Scholar
  15. 15.
    Lemaire, R., Desmons, A., Tabet, J. C., Day, R., Salzet, M., and Fournier, I. (2007) Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections. J Proteome Res. 6, 1295–305.PubMedCrossRefGoogle Scholar
  16. 16.
    Ronci, M., Bonanno, E., Colantoni, A., Pieroni, L., Di, I. C., Spagnoli, L. G., et al. (2008) Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS investigations. Proteomics 8, 3702–14.PubMedCrossRefGoogle Scholar
  17. 17.
    Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 68, 850–8.PubMedCrossRefGoogle Scholar
  18. 18.
    Bairoch, A., Apweiler, R., Wu, C. H., Barker, W. C., Boeckmann, B., Ferro, S., et al. (2005) The universal protein resource (UniProt). Nucleic Acids Res. 33, D154–9.PubMedCrossRefGoogle Scholar
  19. 19.
    Zeeberg, B. R., Feng, W., Wang, G., Wang, M. D., Fojo, A. T., Sunshine, M., et al. (2003) GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol. 4, R28.PubMedCrossRefGoogle Scholar
  20. 20.
    Fowler, C. B., O’Leary, T. J., and Mason, J. T. (2008) Modeling formalin fixation and histological processing with bovine ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde-induced cross-links. Lab Invest. 88, 785–91.PubMedCrossRefGoogle Scholar
  21. 21.
    Chu, W. S., Liang, Q., Liu, J., Wei, M. Q., Winters, M., Liotta, L., et al. (2005) A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section. Lab Invest. 85, 1416–28.PubMedCrossRefGoogle Scholar
  22. 22.
    Fowler, C. B., Cunningham, R. E., Waybright, T. J., Blonder, J., Veenstra, T. D., O’Leary, T. J., et al. (2008) Elevated hydrostatic pressure promotes protein recovery from ­formalin-fixed, paraffin-embedded tissue surrogates. Lab Invest. 88, 185–95.PubMedCrossRefGoogle Scholar
  23. 23.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248–54.PubMedCrossRefGoogle Scholar
  24. 24.
    Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem. 150, 76–85.PubMedCrossRefGoogle Scholar
  25. 25.
    Wisniewski, J. R., Zougman, A., Nagaraj, N., and Mann, M. (2009) Universal sample preparation method for proteome analysis. Nat Methods 6, 359–62.PubMedCrossRefGoogle Scholar
  26. 26.
    Elias, J. E., Haas, W., Faherty, B. K., and Gygi, S. P. (2005) Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations. Nat Methods 2, 667–75.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Carol B. Fowler
    • 1
    Email author
  • Timothy J. O’Leary
    • 2
  • Jeffrey T. Mason
    • 1
  1. 1.Armed Forces Institute of PathologyWashingtonUSA
  2. 2.Veterans Health AdministrationWashingtonUSA

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