Mamoun Ahram 1 * Michael J. Flaig 1 John W. Gillespie 2 Paul H. Duray 3 W. Marston Linehan 4 David K. Ornstein 5 Shulan Niu 6 Yingming Zhao 6 Emanuel F. Petricoin III 7 Michael R. Emmert-Buck 1 1 Pathogenetics Unit, National Cancer Institute, NIH, Bethesda, MD, USA 2 Science Applications International Corporation, National Cancer Institute, Bethesda, MD, USA 3 Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, MD, USA 4 Urologic Oncology Branch, National Cancer Institute, NIH, Bethesda, MD, USA 5 Department of Urology, University of North Carolina, Chapel Hill, NC, USA 6 Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX, USA 7 Tissue Proteomics Unit, Center for Biologics, Evaluation and Research, FDA, Bethesda, MD, USA Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications We previously reported that ethanol fixation and paraffin embedding of tissues pro- duce excellent histomorphology and good preservation of macromolecules. Here, we present a detailed evaluation of ethanol-fixed tissues for proteomic initiatives. When proteins were extracted from ethanol-fixed, paraffin-embedded prostate tis- sue, resolved by two-dimensional gel electrophoresis (2-DE), and stained by stan- dard methods, several hundred protein molecules could be detected and success- fully analyzed by mass spectrometry. Protein profiles obtained from ethanol-fixed tissues were highly similar to those observed from frozen tissues, in contrast to the poor protein recovery from formalin-fixed material. The protein content of specific cells that were microdissected from ethanol-fixed tissue sections using laser cap- ture microdissection could also be successfully analyzed by 2-DE. We observed that eosin staining of tissue sections had a detrimental effect on protein separation, whereas hematoxylin staining had minimal consequence. In order to illustrate the applicability of ethanol-fixed tissues for proteomic discovery studies, we compared the protein profiles of patient-matched, normal prostatic epithelial cells and invasive adenocarcinoma cells obtained from ethanol-fixed, paraffin-embedded tissues. A number of differentially expressed proteins was discovered and identified by mass spectrometry. Immunohistochemical analyses performed on ethanol-fixed tissue sections were in agreement with the proteomic discovery findings. In light of these results, we conclude that ethanol-fixed tissues can be successfully utilized for proteomic analyses. Keywords: Ethanol fixation / Laser capture microdissection / Prostate cancer / Tissue staining PRO 0379 1 Introduction Molecular profiling, or global measurement of mRNA and protein expression in cells, is anticipated to advance our understanding of physiological and pathological condi- tions. For example, elucidation of the expression patterns of normal cells and their tumor counterparts may desig- nate specific tumor markers and lead to a more accurate classification of tumor types, resulting in better diagnosis and prognosis of diseases. Several studies have recently been reported in which RNA expression patterns were employed to classify diseases according to either their type [1], aggressiveness [2], clinical outcome [3], or mo- lecular circuitry [4, 5]. In parallel, proteomic analysis is being utilized in the molecular profiling field and has led to the identification of differentially expressed proteins in cancer progression [6–9]. Several experimental systems (e.g., cells lines, animal models, etc.) are currently available for molecular profiling studies. Although useful, investigators often encounter significant shortcomings with these approaches. For example, removal of cells from their natural tissue envi- ronment and culturing them in vitro may cause major alterations in gene expression [10, 11]. Cultured cells may not be representative of the true expression patterns that occur in vivo. Therefore, the utilization of primary human tissue samples are likely to be critical in addres- sing many biological questions in the future. Correspondence: Dr. Michael Emmert-Buck, 10 Center Drive, Bldg. 10, Rm. 2A33, Pathogenetics Unit, Laboratory of Pathol- ogy, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA E-mail: mbuck@helix.nih.gov Fax: +1-301-594-7582 Abbreviations: LCM, laser capture microdissection; OBG, octyl b-glucopyranoside; T-PER, tissue-protein extraction reagent Proteomics 2003, 3, 413–421 413 * Current address: Molecular Biosciences, Battelle, Pacific Northwest National Laboratory, P. O. Box 999, Mail Stop K4- 12, Richland, WA 99352, USA  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/0404–413 $17.501.50/0