Skip to main content

Advertisement

SpringerLink
Log in

Assessment of the 2-D Gel-Based Proteomics Application of Clinically Archived Formalin-Fixed Paraffin Embedded Tissues

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Hospital tissue repositories possess a vast and valuable supply of disease samples with matched retrospective clinical information. Detection and characterization of disease biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues will greatly aid the understanding of the diseases mechanisms and help in the development of diagnostic and prognostic markers. In this study, the possibility of using full-length proteins extracted from clinically archived FFPE tissues in two-dimensional (2-D) gel-based proteomics was evaluated. The evaluation was done based on two types of tumor tissues (breast and prostate) and two extraction protocols. The comparison of the 2-D patterns of FFPE extracts obtained by two extraction protocols with the matching frozen tissue extracts showed that only 7–10 % of proteins from frozen tissues can be matched to proteins from FFPE tissues. Most of the spots in the 2-D FFPE’s maps had pl 4–6, while the percentages of proteins with pl above 6 were 3–5 times lower in comparison to the fresh/frozen tissue. Despite the three-fold lower number of the detected spots in FFPE maps compared to matched fresh/frozen maps, 67–78 % of protein spots in FFPE could not be matched to the corresponding spots in the fresh/frozen tissue maps indicating irreversible protein modifications. In conclusion, the inability to completely reverse the cross-linked complexes and overcome protein fragmentation with the present day FFPE extraction methods stands in the way of effective use of these samples in 2-D gel based proteomics studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

2-D:

Two-dimensional

2-D DIGE:

Two-dimensional difference in-gel electrophoresis

2-D PAGE:

Two-dimensional polyacrylamide gel electrophoresis

AR:

Antigen retrieval

CHAPS:

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

DTT:

Dithiothreitol

FFPE:

Formalin-fixed paraffin-embedded

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tris:

Tris(hydroxymethyl)aminomethane

References

  1. Bibikova M, Talantov D, Chudin E, Yeakley JM, Chen J, Doucet D, Wickham E, Atkins D, Barker D, Chee M, Wang Y, Fan JB (2004) Quantitative gene expression profiling in formalin-fixed, paraffin-embedded tissues using universal bead arrays. Am J Pathol 165(5):1799–1807

    Article  CAS  Google Scholar 

  2. Huang WY, Sheehy TM, Moore LE, Hsing AW, Purdue MP (2010) Simultaneous recovery of DNA and RNA from formalin-fixed paraffin-embedded tissue and application in epidemiologic studies. Cancer Epidemiol Biomarkers Prev 19(4):973–977

    Article  CAS  Google Scholar 

  3. Specht K, Richter T, Muller U, Walch A, Werner M, Hofler H (2001) Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am J Pathol 158(2):419–429

    Article  CAS  Google Scholar 

  4. von Weizsacker F, Labeit S, Koch HK, Oehlert W, Gerok W, Blum HE (1991) A simple and rapid method for the detection of RNA in formalin-fixed, paraffin-embedded tissues by PCR amplification. Biochem Biophys Res Commun 174(1):176–180

    Article  Google Scholar 

  5. Blonder J, Veenstra TD (2009) Clinical proteomic applications of formalin-fixed paraffin-embedded tissues. Clin Lab Med 29(1):101–113

    Article  Google Scholar 

  6. Reimel BA, Pan S, May DH, Shaffer SA, Goodlett DR, McIntosh MW, Yerian LM, Bronner MP, Chen R, Brentnall TA (2009) Proteomics on fixed tissue specimens—a review. Curr Proteomics 6(1):63–69

    Article  CAS  Google Scholar 

  7. Metz B, Kersten GF, Hoogerhout P, Brugghe HF, Timmermans HA, de Jong A, Meiring H, ten Hove J, Hennink WE, Crommelin DJ, Jiskoot W (2004) Identification of formaldehyde-induced modifications in proteins: reactions with model peptides. J Biol Chem 279(8):6235–6243

    Article  CAS  Google Scholar 

  8. Toews J, Rogalski JC, Clark TJ, Kast J (2008) Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions. Anal Chim Acta 618(2):168–183

    Article  CAS  Google Scholar 

  9. Toews J, Rogalski JC, Kast J (2010) Accessibility governs the relative reactivity of basic residues in formaldehyde-induced protein modifications. Anal Chim Acta 676(1–2):60–67

    Article  CAS  Google Scholar 

  10. Magdeldin S, Yamamoto T (2012) Toward deciphering proteomes of formalin-fixed paraffin-embedded (FFPE) tissues. Proteomics 12(7):1045–1058

    Article  CAS  Google Scholar 

  11. Tanca A, Pagnozzi D, Addis MF (2012) Setting proteins free: progresses and achievements in proteomics of formalin-fixed, paraffin-embedded tissues. Proteomics Clin Appl 6(1–2):7–21

    Article  CAS  Google Scholar 

  12. Addis MF, Tanca A, Pagnozzi D, Crobu S, Fanciulli G, Cossu-Rocca P, Uzzau S (2009) Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissues. Proteomics 9(15):3815–3823

    Article  CAS  Google Scholar 

  13. Azimzadeh O, Barjaktarovic Z, Aubele M, Calzada-Wack J, Sarioglu H, Atkinson MJ, Tapio S (2010) Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics. J Proteome Res 9(9):4710–4720

    Article  CAS  Google Scholar 

  14. Becker KF, Schott C, Hipp S, Metzger V, Porschewski P, Beck R, Nahrig J, Becker I, Hofler H (2007) Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. J Pathol 211(3):370–378

    Article  CAS  Google Scholar 

  15. Chu WS, Liang Q, Liu J, Wei MQ, Winters M, Liotta L, Sandberg G, Gong M (2005) A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section. Lab Invest 85(11):1416–1428

    Article  CAS  Google Scholar 

  16. Chung JY, Lee SJ, Kris Y, Braunschweig T, Traicoff JL, Hewitt SM (2008) A well-based reverse-phase protein array applicable to extracts from formalin-fixed paraffin-embedded tissue. Proteomics Clin Appl 2(10–11):1539–1547

    Article  CAS  Google Scholar 

  17. Hwang SI, Thumar J, Lundgren DH, Rezaul K, Mayya V, Wu L, Eng J, Wright ME, Han DK (2007) Direct cancer tissue proteomics: a method to identify candidate cancer biomarkers from formalin-fixed paraffin-embedded archival tissues. Oncogene 26(1):65–76

    Article  CAS  Google Scholar 

  18. Ikeda K, Monden T, Kanoh T, Tsujie M, Izawa H, Haba A, Ohnishi T, Sekimoto M, Tomita N, Shiozaki H, Monden M (1998) Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J Histochem Cytochem 46(3):397–403

    Article  CAS  Google Scholar 

  19. Jiang X, Feng S, Tian R, Ye M, Zou H (2007) Development of efficient protein extraction methods for shotgun proteome analysis of formalin-fixed tissues. J Proteome Res 6(3):1038–1047

    Article  CAS  Google Scholar 

  20. Nirmalan NJ, Harnden P, Selby PJ, Banks RE (2009) Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting. J Pathol 217(4):497–506

    Article  CAS  Google Scholar 

  21. Nirmalan NJ, Hughes C, Peng J, McKenna T, Langridge J, Cairns DA, Harnden P, Selby PJ, Banks RE (2011) Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations. J Proteome Res 10(2):896–906

    Article  CAS  Google Scholar 

  22. Palmer-Toy DE, Krastins B, Sarracino DA, Nadol JB Jr, Merchant SN (2005) Efficient method for the proteomic analysis of fixed and embedded tissues. J Proteome Res 4(6):2404–2411

    Article  CAS  Google Scholar 

  23. Rahimi F, Shepherd CE, Halliday GM, Geczy CL, Raftery MJ (2006) Antigen-epitope retrieval to facilitate proteomic analysis of formalin-fixed archival brain tissue. Anal Chem 78(20):7216–7221

    Article  CAS  Google Scholar 

  24. Shi SR, Liu C, Balgley BM, Lee C, Taylor CR (2006) Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry. J Histochem Cytochem 54(6):739–743

    Article  CAS  Google Scholar 

  25. Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39(6):741–748

    Article  CAS  Google Scholar 

  26. Guo T, Wang W, Rudnick PA, Song T, Li J, Zhuang Z, Weil RJ, DeVoe DL, Lee CS, Balgley BM (2007) Proteome analysis of microdissected formalin-fixed and paraffin-embedded tissue specimens. J Histochem Cytochem 55(7):763–772

    Article  CAS  Google Scholar 

  27. Scicchitano MS, Dalmas DA, Boyce RW, Thomas HC, Frazier KS (2009) Protein extraction of formalin-fixed, paraffin-embedded tissue enables robust proteomic profiles by mass spectrometry. J Histochem Cytochem 57(9):849–860

    Article  CAS  Google Scholar 

  28. Sprung RW Jr, Brock JW, Tanksley JP, Li M, Washington MK, Slebos RJ, Liebler DC (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(8):1988–1998

    Article  CAS  Google Scholar 

  29. Ahram M, Flaig MJ, Gillespie JW, Duray PH, Linehan WM, Ornstein DK, Niu S, Zhao Y, Petricoin EF 3rd, Emmert-Buck MR (2003) Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications. Proteomics 3(4):413–421

    Article  CAS  Google Scholar 

  30. Bellet V, Boissiere F, Bibeau F, Desmetz C, Berthe ML, Rochaix P, Maudelonde T, Mange A, Solassol J (2008) Proteomic analysis of RCL2 paraffin-embedded tissues. J Cell Mol Med 12(5B):2027–2036

    Article  CAS  Google Scholar 

  31. Ono A, Kumai T, Koizumi H, Nishikawa H, Kobayashi S, Tadokoro M (2009) Overexpression of heat shock protein 27 in squamous cell carcinoma of the uterine cervix: a proteomic analysis using archival formalin-fixed, paraffin-embedded tissues. Hum Pathol 40(1):41–49

    Article  CAS  Google Scholar 

  32. Addis MF, Tanca A, Pagnozzi D, Rocca S, Uzzau S (2009) 2-D PAGE and MS analysis of proteins from formalin-fixed, paraffin-embedded tissues. Proteomics 9(18):4329–4339

    Article  CAS  Google Scholar 

  33. Tanca A, Pagnozzi D, Falchi G, Tonelli R, Rocca S, Roggio T, Uzzau S, Addis MF (2011) Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues. Proteomics 11(5):1005–1011

    Article  CAS  Google Scholar 

  34. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  35. Wolff C, Schott C, Porschewski P, Reischauer B, Becker KF (2011) Successful protein extraction from over-fixed and long-term stored formalin-fixed tissues. PLoS ONE 6(1):e16353

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Many thanks to Dr. Katerina Kubelka-Sabit and Dr. Vanja Filipovski from Clinical Hospital “Sistina”, Skopje, for providing the fresh and FFPE tissues used in this study.

Ethical standard

The study has been approved by the Ethics Committee of the Macedonian Academy of Sciences and Arts.

Conflict of interest

The authors have declared no conflict of interests.

Author information

Authors and Affiliations

  1. Research Centre for Genetic Engineering and Biotechnology “Georgi D Efremov”, Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000, Skopje, Republic of Macedonia

    Katarina Davalieva, Sanja Kiprijanovska & Momir Polenakovic

Authors
  1. Katarina Davalieva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sanja Kiprijanovska
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Momir Polenakovic
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Katarina Davalieva.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Davalieva, K., Kiprijanovska, S. & Polenakovic, M. Assessment of the 2-D Gel-Based Proteomics Application of Clinically Archived Formalin-Fixed Paraffin Embedded Tissues. Protein J 33, 135–142 (2014). https://doi.org/10.1007/s10930-014-9545-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-014-9545-2

Keywords

Search

Navigation