Abstract
In this chapter, we describe the utility of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) as a proteomics platform for the global detection of expressed proteins in formalin-fixed paraffin-embedded (FFPE) tissues and its use for biomarker discovery/identification of proteins that may contribute to cancer development and progression. Formalin fixation and paraffin embedding of tissue is the standard processing methodology practiced in pathology laboratories worldwide, resulting in a highly stable form of tissue that is easily stored due to its inherent stability at room temperature. Consequently, FFPE tissues represent an attractive reservoir of clinical material for conducting retrospective protein biomarker analysis. A limitation for proteomics research in this type of clinical sample is the amount of viable protein that can be obtained from fixed tissues. Tissue biopsies are precious samples that can generally be acquired in very small amounts due to the invasive nature of the sample collection, mainly during surgery or biopsy. Subsequently, the amount of extracted protein can be, in many cases, very limited. The saturation 2D-DIGE technology has emerged as a useful method for protein analysis where only scarce amounts of protein are available. This approach can be adapted successfully to label low-level protein isolated from FFPE tissue.
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References
Kondo T, Seike M, Mori Y, Fujii K, Yamada T, Hirohashi S (2003) Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool. Proteomics 3:1758–1766. https://doi.org/10.1002/pmic.200300531
de la Cuesta F, Alvarez-Llamas G, Maroto AS, Donado A, Juarez-Tosina R, Rodriguez-Padial L, Pinto AG, Barderas MG, Vivanco F (2009) An optimum method designed for 2-D DIGE analysis of human arterial intima and media layers isolated by laser microdissection. Proteomics Clin Appl 3:1174–1184. https://doi.org/10.1002/prca.200900053
Sawhney S, Stubbs R, Hood K (2009) Reproducibility, sensitivity and compatibility of the ProteoExtract subcellular fractionation kit with saturation labeling of laser microdissected tissues. Proteomics 9:4087–4092. https://doi.org/10.1002/pmic.200800949
Arnold GJ, Fröhlich T (2012) 2D DIGE saturation labeling for minute sample amounts. Methods Mol Biol 854:89–112. https://doi.org/10.1007/978-1-61779-573-2_7
Villar M, Torina A, Nuñez Y, Zivkovic Z, Marina A, Alongi A, Scimeca S, La Barbera G, Caracappa S, Vázquez J, Fuente JL (2010) Application of highly sensitive saturation labeling to the analysis of differential protein expression in infected ticks from limited samples. Proteome Sci 8:43. https://doi.org/10.1186/1477-5956-8-43
McNamara LE, Kantawong FA, Dalby MJ, Riehle MO, Burchmore R (2011) Preventing and troubleshooting artefacts in saturation labelled fluorescence 2-D difference gel electrophoresis (saturation DiGE). Proteomics 11:4610–4621. https://doi.org/10.1002/pmic.201100135
Friedman DB, Lilley KS (2008) Optimizing the difference gel electrophoresis (DIGE) technology. Methods Mol Biol 428:93–124
Greengauz-Roberts O, Stöppler H, Nomura S, Yamaguchi H, Goldenring JR, Podolsky RH, Lee JR, Dynan WS (2005) Saturation labeling with cysteine-reactive cyanine fluorescent dyes provides increased sensitivity for protein expression profiling of laser-microdissected clinical specimens. Proteomics 5:1746–1757. https://doi.org/10.1002/pmic.200401068
Weiland F, Lokman NA, Klingler-Hoffmann M, Jobling T, Stephens AN, Sundfeldt K, Hoffmann P, Oehler MK (2020) Ovarian blood sampling identifies junction plakoglobin as a novel biomarker of early ovarian cancer. Front Oncol 10:1767. https://doi.org/10.3389/fonc.2020.01767
Kirana C, Peng L, Miller R, Keating JP, Glenn C, Shi H, Jordan TW, Maddern GJ, Stubbs RS (2019) Combination of laser microdissection, 2D-DIGE and MALDI-TOF MS to identify protein biomarkers to predict colorectal cancer spread. Clin Proteomics 16:3. https://doi.org/10.1186/s12014-019-9223-7
Magdeldin S, Yamamoto T (2012) Toward deciphering proteomes of formalin-fixed paraffin-embedded (FFPE) tissues. Proteomics 12:1045–1058. https://doi.org/10.1002/pmic.201100550
Klopfleisch R, Weiss AT, Gruber AD (2011) Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues. Histol Histopathol 26:797–810
Giusti L, Lucacchini A (2013) Proteomic studies of formalin-fixed paraffin-embedded tissues. Expert Rev Proteomics 10:165–177. https://doi.org/10.1586/epr.13.3
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Dowling, P. (2023). DIGE Saturation Labeling for Scarce Amounts of Protein from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue. In: Ohlendieck, K. (eds) Difference Gel Electrophoresis. Methods in Molecular Biology, vol 2596. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2831-7_9
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DOI: https://doi.org/10.1007/978-1-0716-2831-7_9
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