An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays

Proteome Science

Dimitra Tsavachidou⁵  Bryan T Hennessy¹  Yiling Lu²  Gordon B Mills²  Eric Jonasch⁵  Pheroze Tamboli⁴  Zhenlin Ju³  Wenbin Liu³  Huifang Guo² 

[1] Department of Medical Oncology, Beaumont Hospital, Royal College of Surgeons of Ireland, Dublin, Ireland;Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA;Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA;Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA;Department of Genitourinary Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

关键词: Renal cancer;    Breast cancer;    Reverse phase protein array;    Protein extraction;    Paraffin-embedded tissue;    Formalin-fixed;   

Others  :  817177
DOI  :  10.1186/1477-5956-10-56

 received in 2012-02-17, accepted in 2012-09-10,  发布年份 2012

【 摘 要 】

Introduction

Protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA) is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA.

Results

Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples.

The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p < 0.05) in at least one cell or tissue type, with only 23 markers common in all three sample sets. In addition, FFPE preparations yielded biologically meaningful observations related to pathway signaling status in cell lines, and classification of renal tissues.

Conclusions

With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type.

【 授权许可】

   
2012 Guo et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Lim MS, Elenitoba-Johnson KS: Proteomics in pathology research. Lab Invest 2004, 84(10):1227-1244.
• [2]Crockett DK, Lin Z, Vaughn CP, Lim MS, Elenitoba-Johnson KS: Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS. Lab Invest 2005, 85(11):1405-1415.
• [3]Addis MF, Tanca A, Pagnozzi D, Crobu S, Fanciulli G, Cossu-Rocca P, Uzzau S: Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissues. Proteomics 2009, 9(15):3815-3823.
• [4]Hood BL, Darfler MM, Guiel TG, Furusato B, Lucas DA, Ringeisen BR, Sesterhenn IA, Conrads TP, Veenstra TD, Krizman DB: Proteomic analysis of formalin-fixed prostate cancer tissue. Mol Cell Proteomics 2005, 4(11):1741-1753.
• [5]Shi SR, Liu C, Balgley BM, Lee C, Taylor CR: Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry. J Histochem Cytochem 2006, 54(6):739-743.
• [6]Palmer-Toy DE, Krastins B, Sarracino DA, Nadol JB Jr, Merchant SN: Efficient method for the proteomic analysis of fixed and embedded tissues. J Proteome Res 2005, 4(6):2404-2411.
• [7]Guo T, Wang W, Rudnick PA, Song T, Li J, Zhuang Z, Weil RJ, DeVoe DL, Lee CS, Balgley BM: Proteome analysis of microdissected formalin-fixed and paraffin-embedded tissue specimens. J Histochem Cytochem 2007, 55(7):763-772.
• [8]Hwang SI, Thumar J, Lundgren DH, Rezaul K, Mayya V, Wu L, Eng J, Wright ME, Han DK: Direct cancer tissue proteomics: a method to identify candidate cancer biomarkers from formalin-fixed paraffin-embedded archival tissues. Oncogene 2007, 26(1):65-76.
• [9]Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB, Kornblau SM: Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells. Mol Cancer Ther 2006, 5(10):2512-2521.
• [10]Park ES, Rabinovsky R, Carey M, Hennessy BT, Agarwal R, Liu W, Ju Z, Deng W, Lu Y, Woo HG, et al.: Integrative analysis of proteomic signatures, mutations, and drug responsiveness in the NCI 60 cancer cell line set. Mol Cancer Ther 2010, 9(2):257-267.
• [11]Carey MS, Agarwal R, Gilks B, Swenerton K, Kalloger S, Santos J, Ju Z, Lu Y, Zhang F, Coombes KR, et al.: Functional proteomic analysis of advanced serous ovarian cancer using reverse phase protein array: TGF-beta pathway signaling indicates response to primary chemotherapy. Clin Cancer Res 2010, 16(10):2852-2860.
• [12]Sheehan KM, Calvert VS, Kay EW, Lu Y, Fishman D, Espina V, Aquino J, Speer R, Araujo R, Mills GB, et al.: Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma. Mol Cell Proteomics 2005, 4(4):346-355.
• [13]Spurrier B, Honkanen P, Holway A, Kumamoto K, Terashima M, Takenoshita S, Wakabayashi G, Austin J, Nishizuka S: Protein and lysate array technologies in cancer research. Biotechnol Adv 2008, 26(4):361-369.
• [14]Speer R, Wulfkuhle J, Espina V, Aurajo R, Edmiston KH, Liotta LA, Petricoin EF 3rd: Development of reverse phase protein microarrays for clinical applications and patient-tailored therapy. Cancer Genomics Proteomics 2007, 4(3):157-164.
• [15]Hennessy BT, Lu Y, Gonzalez-Angulo AM, Carey MS, Myhre S, Ju Z, Davies MA, Liu W, Coombes K, Meric-Bernstam F, et al.: A Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers. Clin Proteomics 2010, 6(4):129-151.
• [16]Fowler CB, Cunningham RE, O'Leary TJ, Mason JT: 'Tissue surrogates' as a model for archival formalin-fixed paraffin-embedded tissues. Lab Invest 2007, 87(8):836-846.
• [17]Shi SR, Cote RJ, Taylor CR: Antigen retrieval immunohistochemistry and molecular morphology in the year 2001. Appl Immunohistochem Mol Morphol 2001, 9(2):107-116.
• [18]Chung JY, Lee SJ, Kris Y, Braunschweig T, Traicoff JL, Hewitt SM: A well-based reverse-phase protein array applicable to extracts from formalin-fixed paraffin-embedded tissue. Proteomics Clin Appl 2008, 2:1539-1547.
• [19]Shi SR, Key ME, Kalra KL: 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 1991, 39(6):741-748.
• [20]Shi SR, Imam SA, Young L, Cote RJ, Taylor CR: Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 1995, 43(2):193-201.
• [21]Fowler CB, Cunningham RE, Waybright TJ, Blonder J, Veenstra TD, O'Leary TJ, Mason JT: Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates. Lab Invest 2008, 88(2):185-195.
• [22]Zien A, Aigner T, Zimmer R, Lengauer T: Centralization: a new method for the normalization of gene expression data. Bioinformatics 2001, 17(Suppl 1):S323-S331.
• [23]Wolff C, Schott C, Porschewski P, Reischauer B, Becker KF: Successful protein extraction from over-fixed and long-term stored formalin-fixed tissues. PLoS One 2011, 6(1):e16353.
• [24]Becker KF, Schott C, Hipp S, Metzger V, Porschewski P, Beck R, Nahrig J, Becker I, Hofler H: Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. J Pathol 2007, 211(3):370-378.
• [25]Berg D, Langer R, Tran K, Walch A, Schuster T, Bronger H, Becker KF: Protein Microarray-based Comparison of HER2, Estrogen Receptor, and Progesterone Receptor Status in Core Biopsies and Surgical Specimens From FFPE Breast Cancer Tissues. Appl Immunohistochem Mol Morphol 2011, 19(4):300-305.
• [26]Berg D, Wolff C, Malinowsky K, Tran K, Walch A, Bronger H, Schuster T, Hofler H, Becker KF: Profiling signalling pathways in formalin-fixed and paraffin-embedded breast cancer tissues reveals cross-talk between EGFR, HER2, HER3 and uPAR. J Cell Physiol 2012, 227(1):204-212.
• [27]Wolff C, Malinowsky K, Berg D, Schragner K, Schuster T, Walch A, Bronger H, Hofler H, Becker KF: Signalling networks associated with urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in breast cancer tissues: new insights from protein microarray analysis. J Pathol 2011, 223(1):54-63.
• [28]Silvestri A, Colombatti A, Calvert VS, Deng J, Mammano E, Belluco C, De Marchi F, Nitti D, Liotta LA, Petricoin EF, et al.: Protein pathway biomarker analysis of human cancer reveals requirement for upfront cellular-enrichment processing. Lab Invest 2010, 90(5):787-796.
• [29]Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, et al.: Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012, 366(10):883-892.
• [30]Westermeier R: Sensitive, quantitative, and fast modifications for Coomassie Blue staining of polyacrylamide gels. Proteomics 2006, 6(Suppl 2):61-64.
• [31]Hennessy BT, Lu Y, Poradosu E, Yu Q, Yu S, Hall H, Carey MS, Ravoori M, Gonzalez-Angulo AM, Birch R, et al.: Pharmacodynamic markers of perifosine efficacy. Clin Cancer Res 2007, 13(24):7421-7431.
• [32]Stemke-Hale K, Gonzalez-Angulo AM, Lluch A, Neve RM, Kuo WL, Davies M, Carey M, Hu Z, Guan Y, Sahin A, et al.: An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res 2008, 68(15):6084-6091.
• [33]Hu J, He X, Baggerly KA, Coombes KR, Hennessy BT, Mills GB: Non-parametric quantification of protein lysate arrays. Bioinformatics 2007, 23(15):1986-1994.
• [34]Quackenbush J: Microarray data normalization and transformation. Nat Genet 2002, 32(Suppl):496-501.
• [35]Team RDC: R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; 2010:409.