Abstract
In the functional proteome era, the proteomic profiling of clinicopathologic annotated tissues is an essential step for mining and evaluations of candidate biomarkers for disease. Previously, application of routine proteomic methodologies to clinical tissue specimens has provided unsatisfactory results. Multiplex tissue immunoblotting is a method of transferring proteins from a formalin-fixed, paraffin-embedded tissue section to a stack of membranes which can be applied to a conventional immunoblotting method. A single tissue section can be transferred to up to ten membranes, each of which is probed with antibodies and detected with fluorescent tags. By this approach, total protein and target signals can be simultaneously determined on each membrane; hence each antibody is internally normalized. Phosphorylation specific antibodies as well as antibodies that do not readily work well with paraffin-embedded tissue are applicable to the membranes, expanding the menu of antibodies that can be utilized with formalin-fixed tissue. This novel platform can provide quantitative detection retaining histomorphologic detail in clinical samples and has great potential to facilitate discovery and development of new diagnostic assays and therapeutic agents.
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References
Chuaqui RF, Bonner RF, Best CJ, Gillespie JW, Flaig MJ, Hewitt SM et al (2002) Post-analysis follow-up and validation of microarray experiments. Nat Genet 32:509–514
Hewitt SM, Dear J, Star RA (2004) Discovery of protein biomarkers for renal diseases. J Am Soc Nephrol 15:1677–1689
Simone NL, Paweletz CP, Charboneau L, Petricoin EF, Liotta LA (2000) Laser capture microdissection: beyond functional genomics to proteomics. Mol Diagn 5:301–307
Ornstein DK, Englert C, Gillespie JW, Paweletz CP, Linehan WM, Emmert-Buck MR et al (2000) Characterization of intracellular prostate-specific antigen from laser capture microdissected benign and malignant prostatic epithelium. Clin Cancer Res 6:353–356
Craven RA, Totty N, Harnden P, Selby PJ, Banks RE (2002) Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations. Am J Pathol 160:815–822
Camp RL, Chung GG, Rimm DL (2002) Automated subcellular localization and quantification of protein expression in tissue microarrays. Nat Med 11:1323–1327
Liotta LA, Petricoin E (2000) Molecular profiling of human cancer. Nat Rev Genet 1:48–56
Miyaji T, Hewitt SM, Liotta LA, Star RA (2002) Frozen protein arrays: a new method for arraying and detecting recombinant native tissue proteins. Proteomics 2:1489–1493
Lopez MF (2000) Better approaches to finding the needle in a haystack: optimizing proteome analysis through automation. Electrophoresis 21:1082–1093
Molloy MP, Phadke ND, Maddock JR, Andrews PC (2001) Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins. Electrophoresis 22:1686–1696
Chung JY, Braunschweig T, Baibakov G, Galperin M, Ramesh A, Skacel M et al (2006) Transfer and multiplex immunoblotting of a paraffin embedded tissue. Proteomics 6:767–774
Chung JY, Braunschweig T, Hu N, Roth M, Traicoff JL, Wang QH et al (2006) Profiling of biomarkers in the normal to tumor transition zone of esophageal squamous cell carcinoma by multiplex tissue immunoblotting. Cancer Epidemiol Biomarkers Prev 15:1403–1408
Chung JY, Braunschweig T, Tuttle K, Hewitt SM (2007) Tissue microarrays as a platform for proteomic investigation. J Mol Histol 38:123–128
Traicoff JL, Chung JY, Braunschweig T, Mazo I, Shu Y, Ramesh A et al (2007) Expression of EIF3-p48/INT6, TID1 and Patched in cancer, a profiling of multiple tumor types and correlation of expression. J Biomed Sci 14:395–405
Chung JY, Hong SM, Choi BY, Cho HJ, Yu ES, Hewitt SM (2009) The expression of phospho-AKT, phospho-mTOR, and PTEN in extrahepatic cholangiocarcinoma. Clin Cancer Res 15:660–667
Chung JY, Braunschweig T, Hewitt SM (2006) Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue. Diagn Mol Pathol 15:229–236
Acknowledgment
The authors wish to thank Michael R. Emmert-Buck for his advice and insights into applications for this platform.
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Chung, JY., Hewitt, S.M. (2015). Proteomic Expressional Profiling of a Paraffin-Embedded Tissue by Multiplex Tissue Immunoblotting. 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_21
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DOI: https://doi.org/10.1007/978-1-4939-2694-7_21
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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