Abstract
Biomarkers from tissue-based proteomic studies directly contribute to defining disease states as well as promise to improve early detection or provide for further targeted therapeutics. In the clinical setting, tissue samples are preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks for histological examination. However, proteomic analysis of FFPE tissue is complicated due to the high level of covalently cross-linked proteins arising from formalin fixation. To address these challenges, we developed well-based reverse-phase protein array (RPPA). This approach is a robust protein isolation methodology (29.44 ± 7.8 μg per 1 mm3 of FFPE tissue) paired with a novel on electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots, demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, dried up and vacuum-sealed, remain stable up to 2 months at room temperature. This methodology is directly applicable to FFPE tissue and presents the direct opportunity of addressing hypothesis within clinical trials and well-annotated clinical tissue repositories.
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References
Mason J, O’Leary TJ (1991) Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J Histochem Cytochem 39:225–229
Ikeda K, Monden T, Kanoh T, Tsujie M, Izawa H, Haba A et al (1998) Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J Histochem Cytochem 46:397–403
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 54:739–743
Chu WS, Liang Q, Liu J, Wei MQ, Winters M, Liotta L et al (2005) A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section. Lab Invest 85:1416–1428
Prieto DA, Hood BL, Darfler MM, Guiel TG, Lucas DA, Conrads TP et al (2005) Liquid Tissue™: proteomic profiling of formalin-fixed tissues. Biotechniques 38:S32–S35
Yamashita S, Okada Y (2005) Mechanisms of heat-induced antigen retrieval: analyses in vitro employing SDS-PAGE and immunohistochemistry. J Histochem Cytochem 53:13–21
Addis MF, Tanca A, Pagnozzi D, Crobu S, Fanciulli G, Cossu-Rocca P et al (2009) Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissues. Proteomics 9:3815–3823
Becker KF, Schott C, Hipp S, Metzger V, Porschewski P, Beck R et al (2007) Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. J Pathol 211:370–378
Hultschig C, Kreutzberger J, Seitz H, Konthur Z, Büssow K, Lehrach H (2006) Recent advances of protein microarrays. Curr Opin Chem Biol 10:4–10
Lueking A, Cahill DJ, Müllner S (2005) Protein biochips: a new and versatile platform technology for molecular medicine. Drug Discov Today 10:789–794
Wingren C, Borrebaeck CA (2008) Antibody microarray analysis of directly labeled complex proteomes. Curr Opin Chem Biol 19:55–61
Chung JY, Lee SJ, Ylaya K, 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 2:1539–1547
Chung JY, Braunschweig T, Hong SM, Kwon DS, Eo SH, Cho HJ et al (2014) Assessment of vascular endothelial growth factor in formalin fixed, paraffin embedded colon cancer specimens by means of a well-based reverse phase protein array. Proteome Sci 12:27
Xie R, Chung JY, Ylaya K, Williams RL, Guerrero N, Nakatsuka N et al (2011) Factors influencing the degradation of archival formalin-fixed paraffin-embedded tissue sections. J Histochem Cytochem 59:356–365
Chung JY, Braunschweig T, Hewitt SM (2006) Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue. Diagn Mol Pathol 15:229–236
Acknowledgments
We thank Kris Ylaya for technical assistance. This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
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Chung, JY., Hewitt, S.M. (2015). A Well-Based Reverse-Phase Protein Array of Formalin-Fixed Paraffin-Embedded Tissue. In: Kurien, B., Scofield, R. (eds) Western Blotting. Methods in Molecular Biology, vol 1312. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2694-7_17
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DOI: https://doi.org/10.1007/978-1-4939-2694-7_17
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2693-0
Online ISBN: 978-1-4939-2694-7
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